CiteAb

Fig.2.E showing Western Blotting from the publication: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer.

Fig.8.B showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.6.C showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.6.A showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.5.C showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.5.A showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.5.B showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.6.E showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Fig.2.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: 27-Hydroxycholesterol represses G9a expression via oestrogen receptor alpha in breast cancer.

Fig.7.A showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Neurons and glial cells acquire a senescent signature after repeated mild traumatic brain injury in a sex-dependent manner.

Fig.7.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Neurons and glial cells acquire a senescent signature after repeated mild traumatic brain injury in a sex-dependent manner.

Fig.7.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Neurons and glial cells acquire a senescent signature after repeated mild traumatic brain injury in a sex-dependent manner.

Fig.4.A showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Neurons and glial cells acquire a senescent signature after repeated mild traumatic brain injury in a sex-dependent manner.

Fig.3.A showing Western Blotting from the publication: Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Fig.3.B showing Western Blotting from the publication: Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Fig.4.A showing Western Blotting from the publication: Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Fig.5.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: MicroRNA-145-5p modulates Krüppel-like factor 5 and inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma.

Fig.2.C showing Western Blotting from the publication: Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction.

Fig.2.D showing Western Blotting from the publication: Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction.

Fig.2.A showing Western Blotting from the publication: Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

Fig.1.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

Fig.4.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

Fig.3.A showing Western Blotting from the publication: Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

Fig.3.A showing Western Blotting in a Chlorocebus aethiops (Grivet monkey) sample from the publication: Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

Fig.7.F showing Western Blotting from the publication: CSB promoter downregulation via histone H3 hypoacetylation is an early determinant of replicative senescence.

67 images found

Resistance training suppresses accumulation of senescent fibro-adipogenic progenitors and senescence-associated secretory phenotype in aging rat skeletal muscle.

In GeroScience on 1 April 2025 by Hung, Y. L., Sato, A., et al.

Accumulation of senescent cells in tissues contributes to multiple aging-related pathologies. Senescent fibro-adipogenic progenitors (FAPs) contribute to aging-related muscle atrophy. Resistance training can help to maintain skeletal muscle mass, improve mobility, and reduce certain health risks commonly associated with aging. We investigated, using rat model, the impact of resistance training on FAPs in aging skeletal muscle, which remains unclear. Twenty-two-month-old female rats were divided into sedentary and training groups. The training group rodents were trained to climb a ladder while bearing a load for 20 training sessions over 2 months, after which, the flexor hallucis longus muscles were collected and analyzed. Senescent cells were identified using a senescence-associated β-galactosidase stain and p21 immunohistochemistry (IHC), and FAPs were identified using platelet-derived growth factor receptor alpha IHC. The results indicate that resistance training in rats prevented aging-associated skeletal muscle atrophy and suppressed M2 polarization of macrophages. The number of senescent cells was significantly reduced in the 24-month-old training group, with most of them being FAPs. Conversely, the number of senescent FAPs increased significantly in the 24-month-old sedentary group compared with that in the 18-month-old sedentary group. The number of senescent FAPs in the 24-month-old training group decreased significantly. Resistance training also suppressed the senescence-associated secretory phenotype (SASP). The killer T cell-specific marker, CD8α, was elevated in the skeletal muscles of the aging rats following resistance training, indicating upregulation of recognition and elimination of senescent cells. Overall, resistance training suppressed the accumulation of senescent FAPs and acquisition of SASP in aging skeletal muscles.
© 2024. The Author(s).

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p53-loss induced prostatic epithelial cell plasticity and invasion is driven by a crosstalk with the tumor microenvironment.

In Cell Death & Disease on 26 January 2025 by Yanushko, D., German, B., et al.

Prostate cancer is a heterogeneous disease with a slow progression and a highly variable clinical outcome. The tumor suppressor genes PTEN and TP53 are frequently mutated in prostate cancer and are predictive of early metastatic dissemination and unfavorable patient outcomes. The progression of solid tumors to metastasis is often associated with increased cell plasticity, but the complex events underlying TP53-loss-induced disease aggressiveness remain incompletely understood. Using genetically engineered mice, we show that Trp53 deficiency in Pten-null prostatic epithelial cells (PECs) does not impact early cell proliferation and neoplasia formation, nor growth arrest and senescence entry at a later time. However, Trp53-deficiency enhances invasive adenocarcinoma development and promotes metastatic cell dissemination. Importantly, our single-cell transcriptomic and chromatin accessibility analyses combined with histological examinations uncovered an epithelial cell population characterized by an induction of Jak/Stat3 signaling and displaying mesenchymal features. Moreover, we show that the transcriptomic signature of this cell population is prominent in tumors of patients with high-risk prostate cancer or metastatic disease. In addition, our in vivo and organoid-based experiments provide evidence that PEC plasticity occurs through bi-directional communication with cancer-associated fibroblasts (CAFs). Thus, our study demonstrates that p53 loss induces a protumorigenic crosstalk between PECs and CAFs, and identifies new vulnerabilities that might be targeted to limit cancer progression.
© 2025. The Author(s).

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Cold exposure reinstates NAD+ levels and attenuates hepatocellular carcinoma.

In Cell Stress on 9 January 2025 by Grazioso, T. P., Del Mar Rigual, M., et al.

Cold exposure has been historically used for medicinal purposes, but its benefits and associated mechanisms in mammalian organisms still remain unclear. Here, we explore the chemoprotective properties of cold temperature using a mouse model of hepatocellular carcinoma (HCC) that recapitulates several human features. Chronic cold exposure is shown to prolong lifespan in diseased mice, enhance liver health, and suppress the development of aggressive HCC, preventing hepatocellular hypertrophy, high-grade oval cell hyperplasia, liver steatosis, and aberrant hepatocyte hyperproliferation. Mechanistically, exposure to cold temperatures reinstates NAD+ levels in the HCC mouse models that originally exhibited low NAD+ levels, a contributing process to the development of liver tumors. These findings uncover the role of cold therapy to attenuate HCC development and potentially other existing malignancies involving NAD+ modulation.

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Synergistic rescue of temperature-sensitive p53 mutants by hypothermia and arsenic trioxide.

In Molecular Carcinogenesis on 1 November 2024 by Lu, J., Chen, L., et al.

The p53 tumor suppressor is inactivated by mutations in about 50% of tumors. Rescuing the transcriptional function of mutant p53 has potential therapeutic benefits. Approximately 15% of p53 mutants are temperature sensitive (TS) and regain maximal activity at 32°C. Proof of concept study showed that induction of 32°C hypothermia in mice restored TS mutant p53 activity and inhibited tumor growth. However, 32°C is the lower limit of therapeutic hypothermia procedures for humans. Higher temperatures are preferable but result in suboptimal TS p53 activation. Recently, arsenic trioxide (ATO) was shown to rescue the conformation of p53 structural mutants by stabilizing the DNA binding domain. We examined the responses of 17 frequently observed p53 TS mutants to functional rescue by temperature shift and ATO. The results showed that ATO only rescued mild p53 TS mutants with high basal activity at 37°C. Mild TS mutants showed a common feature of regaining significant activity at the semi-permissive temperature of 35°C and could be further stimulated by ATO at 35°C. TS p53 rescue by ATO was antagonized by the cellular redox mechanism and was rapidly reversible. Inhibition of glutathione (GSH) biosynthesis enhanced ATO rescue efficiency and sustained p53 activity after ATO washout. The results suggest that mild TS p53 mutants are uniquely responsive to functional rescue by ATO due to small thermostability deficits and inherent potential to regain active conformation. Combining mild hypothermia and ATO may provide an effective and safe procedure for targeting tumors with p53 TS mutations.
© 2024 Wiley Periodicals LLC.

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Transient proliferation by reversible YAP and mitogen-control of the cyclin D1/p27 ratio

Preprint on BioRxiv : the Preprint Server for Biology on 13 October 2024 by Ferrick, K. R., Fan, Y., et al.

ABSTRACT Hippo-YAP signaling orchestrates epithelial tissue repair and is therefore an attractive target in regenerative medicine. Yet it is unresolved how YAP integrates with mitogen signaling and contact inhibition to control the underlying transient proliferative response. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle. YAP increases this ratio indirectly, in concert with mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Increasing YAP activity by ablating the suppressor Merlin/NF2 reveals a balancing mechanism in which YAP suppression and contact inhibition of proliferation can be recovered but only at higher local cell density. Thus, critical for tissue repair, robust proliferation responses result from the YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is only reversed by delayed suppression of receptor signaling after contact inhibition of YAP.

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ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer.

In EMBO Mol Med on 1 October 2024 by Pozzato, C., Outeiro-Pinho, G., et al.

Fig.2.E

Fig.2.E showing Western Blotting from the publication: ERK5 suppression overcomes FAK inhibitor resistance in mutant KRAS-driven non-small cell lung cancer.

ERK5 and CDK5 synergistically promote FAK function.(A) Percentage of proliferation inhibition of A549, A427 and H460 cell lines treated with increasing doses of XMD8-92 or Seliciclib or in combination (top) 48 h after treatment and representative crystal violet-stained cells 72 h after drug treat... more

ERK5 and CDK5 synergistically promote FAK function.(A) Percentage of proliferation inhibition of A549, A427 and H460 cell lines treated with increasing doses of XMD8-92 or Seliciclib or in combination (top) 48 h after treatment and representative crystal violet-stained cells 72 h after drug treatment (bottom). The combination index (CI) showing the synergistic effect of combination of the 2 drugs is indicated; n = 3. (B–D) Relative quantification of Annexin V (AV) + Annexin V/PI (AV/PI)-positive cells by flow cytometry; n = 4 (B), colony forming capacity; n = 3 (C) and percentage of cell migration; n = 10 (D) of A549 cells treated with 10 µM XMD8-92 or Seliciclib or in combination, except for the colony formation assay in which cells were treated with 5 µM of each drug. (E) Immunoblot analysis of the indicated targets in A549 cells treated with the indicated doses of Seliciclib or XMD8-92 or in combination for 24 h. (F) Immunoblot analysis of the indicated targets in A549 cells treated with 10 µM XMD8-92 or 10 µM Seliciclib or in combination for 24 h. (G) Immunoblot analysis for the indicated targets in A549 cells transduced with shRNA control (pLKO.1 hygro + Tet-pLKO-puro) or a shRNA against CDK5 (Tet-pLKO-puro-shCDK5 + pLKO.1 hygro), or a shRNA against ERK5 (pLKO.1 hygro-shERK5 + Tet-pLKO-puro) or in combination (Tet-pLKO-puro-shCDK5 + pLKO.1 hygro-shERK5). After transduction and selection, cells were harvested for protein extraction 72 h after doxycycline (1 μg/mL) induction. (H, I) Relative cell number (H) and Annexin V (AV) + Annexin V/PI (AV/PI)-positive cell quantification by flow cytometry (I) in A549 cells treated as in (G) 72 h after doxycycline (1 μg/mL) induction; n = 3. Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. Source data are available online for this figure. less

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Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

In Cell Death Dis on 29 May 2024 by Neuwahl, J., Neumann, C. A., et al.

Fig.8.B

Fig.8.B showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

Primary senescent fibroblast and epithelial cells are resistant to PI3K-inhibitor-induced senolysis.A The immortalized primary fibroblast BJ/hTERT and epithelial RPE-340/hTERT cell lines were exposed to 20 Gy γIR and senescence induction was determined by staining for senescence-associated beta-g... more

Primary senescent fibroblast and epithelial cells are resistant to PI3K-inhibitor-induced senolysis.A The immortalized primary fibroblast BJ/hTERT and epithelial RPE-340/hTERT cell lines were exposed to 20 Gy γIR and senescence induction was determined by staining for senescence-associated beta-galactosidase activity at day 4 and day 7 following irradiation. Senescent cells display a strong blue color. Microscopic pictures were taken with the same 10x magnification (scale bar 100 µM in the lower right panel) and are representative of at least three independent experiments. B Western blot analyses of the expression of the indicated proteins in BJ/hTERT and RPE-340/hTERT cells at one, three and seven days after 20 Gy γIR. The blots shown are representative of at least three independent experiments. C, D Proliferating and senescent (day 7 after γIR) BJ/hTERT (C) and RPE-340/hTERT (D) cells were exposed to the indicated concentrations of the PI3K inhibitors PX-866 and BAY80-6946, the senolytic compound Navitoclax, and the pan-kinase inhibitor staurosporine (3 µM) as a positive control. After 24 h, LDH activity in the cell culture supernatant as a marker for cell death was determined. The data shown are the mean ± SD of at least three independent experiments. Individual data points are represented in the bar charts by small black squares. Significance indicators relate to the corresponding control (proliferating/senescent) treated with DMSO. *p < 0.05, **p < 0.01, ***p < 0.001. less

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Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

In Cell Death Dis on 29 May 2024 by Neuwahl, J., Neumann, C. A., et al.

Fig.6.C

Fig.6.C showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

PX-866 induces proteasomal degradation of p21 in senescent cells.A, B Proliferating and senescent (day 7 after γIR) HCT116 cells were transfected with a non-targeting control siRNA or siRNAs directed against p53 or p21. 48 h after transfection, the cells were additionally treated with PX-866 (3 µ... more

PX-866 induces proteasomal degradation of p21 in senescent cells.A, B Proliferating and senescent (day 7 after γIR) HCT116 cells were transfected with a non-targeting control siRNA or siRNAs directed against p53 or p21. 48 h after transfection, the cells were additionally treated with PX-866 (3 µM) for another 24 h. Cell death was assessed by quantifying LDH activity released into the cell culture supernatant (B), whereas inhibition of AKT phosphorylation and the knockdown of p53 or p21 protein expression was verified by western blotting (A). C–F Senescent (day 7 after γIR) HCT116 (C, D) or MCF-7 (E, F) cells were treated with either 3 µM PX-866, 10 µM MG-132 or a combination of both. At the indicated times, the cells were harvested and the cell extracts subjected to western blot analysis (C, E). The blots shown are representative of at least three independent experiments. The expression of p21 was quantified densitometrically using the Image Studio (Ver. 5.2) software (LI-COR). Relative p21 levels normalized to the no treatment sample (also indicated by a gray line) were plotted (D, F). Data shown are the mean ± SD of at least three independent experiments. Individual data points are represented in the bar charts by small black squares. ns not significant *p < 0.05, **p < 0.01, ***p < 0.001. less

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Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

In Cell Death Dis on 29 May 2024 by Neuwahl, J., Neumann, C. A., et al.

Fig.6.A

Fig.6.A showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

PX-866 induces proteasomal degradation of p21 in senescent cells.A, B Proliferating and senescent (day 7 after γIR) HCT116 cells were transfected with a non-targeting control siRNA or siRNAs directed against p53 or p21. 48 h after transfection, the cells were additionally treated with PX-866 (3 µ... more

PX-866 induces proteasomal degradation of p21 in senescent cells.A, B Proliferating and senescent (day 7 after γIR) HCT116 cells were transfected with a non-targeting control siRNA or siRNAs directed against p53 or p21. 48 h after transfection, the cells were additionally treated with PX-866 (3 µM) for another 24 h. Cell death was assessed by quantifying LDH activity released into the cell culture supernatant (B), whereas inhibition of AKT phosphorylation and the knockdown of p53 or p21 protein expression was verified by western blotting (A). C–F Senescent (day 7 after γIR) HCT116 (C, D) or MCF-7 (E, F) cells were treated with either 3 µM PX-866, 10 µM MG-132 or a combination of both. At the indicated times, the cells were harvested and the cell extracts subjected to western blot analysis (C, E). The blots shown are representative of at least three independent experiments. The expression of p21 was quantified densitometrically using the Image Studio (Ver. 5.2) software (LI-COR). Relative p21 levels normalized to the no treatment sample (also indicated by a gray line) were plotted (D, F). Data shown are the mean ± SD of at least three independent experiments. Individual data points are represented in the bar charts by small black squares. ns not significant *p < 0.05, **p < 0.01, ***p < 0.001. less

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Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

In Cell Death Dis on 29 May 2024 by Neuwahl, J., Neumann, C. A., et al.

Fig.6.E

Fig.6.E showing Western Blotting from the publication: Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells.

PX-866 induces proteasomal degradation of p21 in senescent cells.A, B Proliferating and senescent (day 7 after γIR) HCT116 cells were transfected with a non-targeting control siRNA or siRNAs directed against p53 or p21. 48 h after transfection, the cells were additionally treated with PX-866 (3 µ... more

PX-866 induces proteasomal degradation of p21 in senescent cells.A, B Proliferating and senescent (day 7 after γIR) HCT116 cells were transfected with a non-targeting control siRNA or siRNAs directed against p53 or p21. 48 h after transfection, the cells were additionally treated with PX-866 (3 µM) for another 24 h. Cell death was assessed by quantifying LDH activity released into the cell culture supernatant (B), whereas inhibition of AKT phosphorylation and the knockdown of p53 or p21 protein expression was verified by western blotting (A). C–F Senescent (day 7 after γIR) HCT116 (C, D) or MCF-7 (E, F) cells were treated with either 3 µM PX-866, 10 µM MG-132 or a combination of both. At the indicated times, the cells were harvested and the cell extracts subjected to western blot analysis (C, E). The blots shown are representative of at least three independent experiments. The expression of p21 was quantified densitometrically using the Image Studio (Ver. 5.2) software (LI-COR). Relative p21 levels normalized to the no treatment sample (also indicated by a gray line) were plotted (D, F). Data shown are the mean ± SD of at least three independent experiments. Individual data points are represented in the bar charts by small black squares. ns not significant *p < 0.05, **p < 0.01, ***p < 0.001. less

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Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

In Commun Biol on 31 October 2022 by Ludikhuize, M. C., Gevers, S., et al.

Fig.3.A

Fig.3.A showing Western Blotting from the publication: Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Inactive p53 leads to 5-FU-induced DNA damage and cell death due to the lack of G1 arrest in TPOs.a Western blot detection of p53, p21 and vinculin in WT and AK organoids treated with 5-FU for 4, 8, 16, 24, and 48 h (blot representative for n = 4). WT and AK were run on the same gel. b Western bl... more

Inactive p53 leads to 5-FU-induced DNA damage and cell death due to the lack of G1 arrest in TPOs.a Western blot detection of p53, p21 and vinculin in WT and AK organoids treated with 5-FU for 4, 8, 16, 24, and 48 h (blot representative for n = 4). WT and AK were run on the same gel. b Western blot detection of p53, p21, (p)Rb and vinculin in WT and CRC organoids treated with 5-FU for 48 h (representative for n = 4). WT, AK, AP, APK, APKS were run together on the same gels. c Cell cycle profile determined by flow cytometry in WT and CRC organoids treated with 5-FU for 48 h (mean ± SEM, n = 4 (WT), 3 (AK), 5 (AP), 7 (APK), 6 (APKS), Kruskal–Wallis test, unpaired t-test and Mann–Whitney test). d Western blot analysis of γH2AX and tubulin of AP, APK and APKS organoids treated with 5-FU for 48 h. Palbociclib treatment was started 24 h before 5-FU treatment to arrest cells in G1 (representative for n = 3). AP, APK, and APKS were run together on the same gels. e, f Representative brightfield imaging (e) and cell viability analysis (f) of AP, APK, and APKS organoids treated with 5-FU for 6 days. Palbociclib treatment started 24 h before 5-FU treatment to arrest cells in G1 (scale bar = 500 µm, mean ± SEM, n = 3, one-way ANOVA, Sidak’s multiple comparisons test). g Western blot detection of p53, p21, γH2AX, and β-actin in doxycycline-inducible APK-P53OE and APKS -P53OE organoids treated with 5-FU for 48 h (doxycycline (40 ng/ml for APK and 200 ng/ml for APKS) treatment was started 16 h before 5-FU administration) (representative for n = 3). APK and APKS were run on separate gels. h Cell cycle profiles of doxycycline-inducible APK-P53OE and APKS-P53OE organoids treated with 5-FU for 48 h (doxycycline (40 ng/ml for APK and 200 ng/ml for APKS) treatment was started 16 h before 5-FU administration) (mean ± SEM, n = 3, one-way ANOVA, Sidak’s multiple comparisons test). i, j Brightfield images (i) and cell viability analysis (j) of doxycycline-inducible APK-P53OE and APKS-P53OE organoids by flow cytometry to distinguish alive (DAPI−) from dead (DAPI+) cells upon 4 days of 5-FU treatment. APK: doxycycline (40 ng/ml) treatment was started 16 h before and washed away 24 h after 5-FU administration. APKS: doxycycline (200 ng/ml) treatment was started 16 h before 5-FU administration and was not washed away (scale bar = 500 µm, mean ± SEM, n = 6 (APK-P53 OE), 4 (APKS-P53 OE), one-way ANOVA, Sidak’s multiple comparisons test). ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. less

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Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

In Commun Biol on 31 October 2022 by Ludikhuize, M. C., Gevers, S., et al.

Fig.3.B

Fig.3.B showing Western Blotting from the publication: Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Inactive p53 leads to 5-FU-induced DNA damage and cell death due to the lack of G1 arrest in TPOs.a Western blot detection of p53, p21 and vinculin in WT and AK organoids treated with 5-FU for 4, 8, 16, 24, and 48 h (blot representative for n = 4). WT and AK were run on the same gel. b Western bl... more

Inactive p53 leads to 5-FU-induced DNA damage and cell death due to the lack of G1 arrest in TPOs.a Western blot detection of p53, p21 and vinculin in WT and AK organoids treated with 5-FU for 4, 8, 16, 24, and 48 h (blot representative for n = 4). WT and AK were run on the same gel. b Western blot detection of p53, p21, (p)Rb and vinculin in WT and CRC organoids treated with 5-FU for 48 h (representative for n = 4). WT, AK, AP, APK, APKS were run together on the same gels. c Cell cycle profile determined by flow cytometry in WT and CRC organoids treated with 5-FU for 48 h (mean ± SEM, n = 4 (WT), 3 (AK), 5 (AP), 7 (APK), 6 (APKS), Kruskal–Wallis test, unpaired t-test and Mann–Whitney test). d Western blot analysis of γH2AX and tubulin of AP, APK and APKS organoids treated with 5-FU for 48 h. Palbociclib treatment was started 24 h before 5-FU treatment to arrest cells in G1 (representative for n = 3). AP, APK, and APKS were run together on the same gels. e, f Representative brightfield imaging (e) and cell viability analysis (f) of AP, APK, and APKS organoids treated with 5-FU for 6 days. Palbociclib treatment started 24 h before 5-FU treatment to arrest cells in G1 (scale bar = 500 µm, mean ± SEM, n = 3, one-way ANOVA, Sidak’s multiple comparisons test). g Western blot detection of p53, p21, γH2AX, and β-actin in doxycycline-inducible APK-P53OE and APKS -P53OE organoids treated with 5-FU for 48 h (doxycycline (40 ng/ml for APK and 200 ng/ml for APKS) treatment was started 16 h before 5-FU administration) (representative for n = 3). APK and APKS were run on separate gels. h Cell cycle profiles of doxycycline-inducible APK-P53OE and APKS-P53OE organoids treated with 5-FU for 48 h (doxycycline (40 ng/ml for APK and 200 ng/ml for APKS) treatment was started 16 h before 5-FU administration) (mean ± SEM, n = 3, one-way ANOVA, Sidak’s multiple comparisons test). i, j Brightfield images (i) and cell viability analysis (j) of doxycycline-inducible APK-P53OE and APKS-P53OE organoids by flow cytometry to distinguish alive (DAPI−) from dead (DAPI+) cells upon 4 days of 5-FU treatment. APK: doxycycline (40 ng/ml) treatment was started 16 h before and washed away 24 h after 5-FU administration. APKS: doxycycline (200 ng/ml) treatment was started 16 h before 5-FU administration and was not washed away (scale bar = 500 µm, mean ± SEM, n = 6 (APK-P53 OE), 4 (APKS-P53 OE), one-way ANOVA, Sidak’s multiple comparisons test). ns: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. less

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Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

In Commun Biol on 31 October 2022 by Ludikhuize, M. C., Gevers, S., et al.

Fig.4.A

Fig.4.A showing Western Blotting from the publication: Rewiring glucose metabolism improves 5-FU efficacy in p53-deficient/KRASG12D glycolytic colorectal tumors.

Inactive p53 leads to 5-FU-induced DNA damage and cell death due to the lack of G1 arrest in PDOs.a Western blot detection of p53, p21 and tubulin in WT and P9t, P16t and P19bt organoids treated with 5-FU for 48 h (representative for n = 3). WT, P9t, P16t and P19bt were run together on the same g... more

Inactive p53 leads to 5-FU-induced DNA damage and cell death due to the lack of G1 arrest in PDOs.a Western blot detection of p53, p21 and tubulin in WT and P9t, P16t and P19bt organoids treated with 5-FU for 48 h (representative for n = 3). WT, P9t, P16t and P19bt were run together on the same gels. b Quantification of cells with DNA damage by flow cytometry of P9t, P16t and P19bt organoids treated with 5-FU for 48 h and stained with anti-γH2AX (mean ± SEM, n = 6 (P9t), 3 (P16t), 4 (P19bt), one-way ANOVA, Sidak’s multiple comparisons test). c Cell cycle profile analysis by flow cytometry of P9t, P16t, and P19bt organoids treated with 5-FU for 48 h and stained with DAPI (mean ± SEM, n = 6 (P9t), 3 (P16t), 4 (P19bt) 4, P9t and P16t: unpaired t-test, P19bt: Mann–Whitney test). d, e Brightfield images (d) and cell viablility analysis (e) of P9t, P16t, and P19bt organoids treated with 5-FU for 6 days (scale bar = 500 µm, mean ± SEM, n = 5 (P9t), 8 (P16t), 4 (P19bt) 4, one-way ANOVA, Sidak’s multiple comparisons test). f, g Western blot detection of p53, p21, γH2AX, and β-actin (f) and cell cycle profile analysis (g) in doxycycline-inducible P16t-P53OE and P19bt-P53OE organoids treated with 5-FU for 48 h. Doxycycline treatment (200 ng/ml) was started 16 h before and washed away 24 h after 5-FU administration (representative for n = 3, mean ± SEM, n = 4 (P16t-P53OE), 3 (P19bt-P53OE), one-way ANOVA, Sidak’s multiple comparisons test). P16t-P53OE and P19bt-P53OE were run together on the same gels. h, i Bright field images (h) and cell viability analysis (i) of doxycycline-inducible P16t-P53OE and P19bt-P53OE organoids treated with 5-FU for 6 days. Doxycycline treatment (200 ng/ml) was started 16 h before and washed away 24 h after 5-FU administration (scale bar = 500 µm, mean ± SEM, n = 5, one-way ANOVA, Sidak’s multiple comparisons test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. less

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MicroRNA-145-5p modulates Krüppel-like factor 5 and inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma.

In BMC Mol Cell Biol on 14 July 2022 by Yuan, C. H., Hsu, W. C., et al.

Fig.5.A

Fig.5.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: MicroRNA-145-5p modulates Krüppel-like factor 5 and inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma.

MiR-145-5p and KLF5 modulates phospho-FAKTyr397 expression in nasopharyngeal carcinoma (NPC) cells. a MiR-145-5p induced p21 and reduced cyclin D1, cyclin B1, pro-caspase 3 and PARP protein expression post-transfection with scramble control or miR-145-5p mimics in NPC-TW03 and NPC-TW04 cells, wer... more

MiR-145-5p and KLF5 modulates phospho-FAKTyr397 expression in nasopharyngeal carcinoma (NPC) cells. a MiR-145-5p induced p21 and reduced cyclin D1, cyclin B1, pro-caspase 3 and PARP protein expression post-transfection with scramble control or miR-145-5p mimics in NPC-TW03 and NPC-TW04 cells, were probes with anti cyclin D1, cyclin B1, p21, pro-caspase 3, PARP and specific antibodies for each compartments corresponding to anti α-tubulin. b MiR-145-5p down-regulates phospho-FAKTyr397 protein expression post-transfection with scramble control or miR-145-5p mimics in NPC-TW03 and NPC-TW04 cells, were probes with anti phospho-FAKTyr397, FAK and specific antibodies for each compartments corresponding to anti β-actin. c Quantification of phospho-FAKTyr397/FAK expression are the average of three independent experiments, using the Image J software and β-actin as a reference. d Western blot analysis indicating phospho-FAKTyr397 and FAK expression in NPC-TW03 and NPC-TW04 cells post-transfection with CMV or CMV-KLF5 vectors, were probes with anti phospho-FAKTyr397, FAK and specific antibodies for each compartments corresponding to anti β-actin. e Quantification of phospho-FAKTyr397/FAK expression are the average of three independent experiments, using the Image J software and β-actin as a reference. f The possible molecular network between KLF5 and FAK was analyzed by Ingenuity Pathway Analysis. g PROMO tool identified the putative transcription factor binding sites in FAK DNA sequences. Data are shown as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, respectively less

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Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

In Cell Death Dis on 27 July 2020 by Makarevich, O., Sabirzhanov, B., et al.

Fig.2.A

Fig.2.A showing Western Blotting from the publication: Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

Mithramycin attenuates etoposide-induced apoptotic pathways downstream of p53 activation.a, c RCN were treated with 50µM etoposide +/−200 nM mithramycin. After 3, 6, 16, or 24 h, cells were harvested. Equal amounts of whole-cell lysates were loaded onto an SDS-polyacrylamide gel and after electro... more

Mithramycin attenuates etoposide-induced apoptotic pathways downstream of p53 activation.a, c RCN were treated with 50µM etoposide +/−200 nM mithramycin. After 3, 6, 16, or 24 h, cells were harvested. Equal amounts of whole-cell lysates were loaded onto an SDS-polyacrylamide gel and after electrophoretic separation and transfer to a membrane were incubated with antibodies against DNA-damage markers: phospho-ATM (S1981) and γH2AX, or phospho-p53 (S15) and its downstream targets: PUMA and p21 (a). Other membranes were incubated with antibodies against cleaved/activated caspase-3 (Casp3) and caspase-7 (Casp7), their substrates α-Fodrin and cleaved PARP, as well as PSD95 (c). Protein levels (of bands indicated by arrows) were quantified by densitometry, normalized to appropriate β-actin signal and are presented as normalized fold change compared with control levels. Representative actin blots are shown here. n = 3/group for all groups. Data represent mean ±SD. Significance assigned based on one-way ANOVA and Tukey post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001, ^^^^p < 0.0001 vs etoposide alone. b Phospho-p53 (S15) levels were also analyzed by immunocytochemistry after 6h. A representative set of × 63 images is shown here, white arrows indicate sample neuron co-localized with ph-p53 (S15). Quantification of cells was done across at least 6 fields per treatment. Data are presented as an unbinned cumulative frequency distribution. Significance assigned based on Kruskal–Wallis test followed by Dunn’s post hoc analysis; ****p < 0.0001 vs control. less

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Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

In Int J Mol Sci on 23 July 2020 by Borkham-Kamphorst, E., Haas, U., et al.

Fig.4.C

Fig.4.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

Lcn2−/− hepatocytes reflecting strongest ER stress responses. (A) Real-time quantitative polymerase chain reaction (RT-qPCR) showing Lcn2 mRNA and ER stress-markers Bip/Grp78, Grp94, Chop, and Creb3L3 upon 24 h incubation with IL-1β, tunicamycin (TM), and thapsigargin (TG). IL-1β significantly in... more

Lcn2−/− hepatocytes reflecting strongest ER stress responses. (A) Real-time quantitative polymerase chain reaction (RT-qPCR) showing Lcn2 mRNA and ER stress-markers Bip/Grp78, Grp94, Chop, and Creb3L3 upon 24 h incubation with IL-1β, tunicamycin (TM), and thapsigargin (TG). IL-1β significantly induced Lcn2 mRNA expression, while Bip/Grp78, Grp94, and Chop mRNA levels were also markedly increased by TM or TG stimulation, while Lcn2−/− hepatocytes showed the highest levels. Additionally, the Lcn2−/− hepatocytes possessed already high basal levels of Creb3L3. Details about the statistics are given in Supplementary Table S6. (B) Semi-quantitative PCR depicting unspliced (Xbp1u) and spliced Xbp1 (Xbp1s) mRNA. Lcn2−/− hepatocytes incubated with TM showing the most significant amounts of Xbp1s. (C) Western blot analysis of ER stress marker proteins GRP94, BiP, IRE1α, ATF6, GADD34, p-eIF2α, eIF2α, ATF4, and CHOP with β-actin as loading controls. Lcn2−/− hepatocytes showed the highest ER stress responses with huge amounts of CHOP, while Chop−/− hepatocytes increased higher baselines of p-eIF2α with decreased GADD34 and other UPR signaling proteins. (D) TM-induced ER stress was showed to activate and cleave CREB3L3/CREBH. TM activated NF-κB p65, TRAF2/JNK/c-Jun, and further inhibited LCN2 glycosylation, as evidenced by shifting of the LCN2 molecular weight, with GAPDH as loading controls. (E) TM altered expression of Bcl2 family proteins, Lcn2−/− hepatocytes were clearly demonstrated to decrease Bcl-xL, and increase Bax, Bak, and PUMA with higher Cytochrome c levels. GAPDH was applied as loading control. less

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Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

In Int J Mol Sci on 23 July 2020 by Sabirzhanov, B., Makarevich, O., et al.

Fig.3.A

Fig.3.A showing Western Blotting from the publication: Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

Ionizing radiation (IR) induces rapid and dose-dependent activation of DNA-damage and p53 pathways in primary cortical neurons. (A) Western blots for Ph-ATM(Ser 1981), γ-H2A.X, p53 and p21. (B) Ph-ATM (Ser1981) (F(6,14) = 246.4, p < 0.0001 for 30 min 2, 8 and 32Gy, compared to control; p = 0.0384... more

Ionizing radiation (IR) induces rapid and dose-dependent activation of DNA-damage and p53 pathways in primary cortical neurons. (A) Western blots for Ph-ATM(Ser 1981), γ-H2A.X, p53 and p21. (B) Ph-ATM (Ser1981) (F(6,14) = 246.4, p < 0.0001 for 30 min 2, 8 and 32Gy, compared to control; p = 0.0384 for 24 h 8Gy, p = 0.0483 for 24 h 32Gy, compared to control; p < 0.0001 for 24 h groups, compared to 30 min and for 30 min 8Gy, compared to 30 min 2Gy. (C) γ-H2A.X (F(6,14) = 47.02, p < 0.0001 for 30 min 8 and 32Gy compared, to control; p < 0.0001 for 30 min 8Gy compared to 30 min 2Gy). (D) γ-H2A.X normalized to total H2A.X (F 6,14) = 102.2, p < 0.0001 for 30 min 8 and 32Gy, compared to control; p < 0.0001 for 30 min 8Gy, compared to 30 min 2Gy; p = 0.045 for 30 min 32Gy, compared to 30 min 8Gy). (E) H2A.X normalized to β-actin. (F) Ph-p53 (F(6,14) = 351.5, p < 0.0001 for 30 min 2, 8 and 32Gy, and for 24 h 8 and 32Gy; p < 0.0001 for 8Gy compared to 2Gy and 32Gy, compared to 8Gy at 30 min; p = 0.0006 for 24 h 8Gy, compared to 24 h 2Gy; p < 0.0001 for 24 h 32Gy, compared to 24 h 8Gy; p < 0.0001 for 24 h 8Gy and 32Gy, compared to control). (G) Ph-p53 normalized to total p53 (F(6,14) = 114.5, p < 0.0001 for 30 min 2, 8 and 32Gy; p = 0.0007 for 24 h 8Gy, p < 0.0001 for 24 h 32Gy, compared to control; p = 0.0016 for 30 min 8Gy, compared to 30 min 2Gy, p < 0.0001 for 30 min 32Gy, compared to 30 min 8Gy; p = 0.0292 for 24 h 8Gy, compared to 24 h 2Gy). (H) p53 normalized to β-actin. (I) p21 (F(6,14) = 32.95, p = 0.0011 for 24 h 2Gy, p < 0.0001 for 24 h 8Gy and p = 0.0001 for 24 h 32Gy, compared to control). Data presented as fold change to non-irradiated control levels after normalization. n = 3/group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. (J) Neurons were collected 30 min and 24 h after 2, 8 and 32Gy irradiation. Total RNA was used for qPCR analysis. qPCR quantification of p21 mRNA. (F(6,14) = 17.7, p < 0.0001 for all 24 h time points, compared to control). Data presented as fold change to non-irradiated control group. n = 3/group; **** p < 0.0001 vs. control. less

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Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

In Sci Rep on 10 February 2020 by López de la Oliva, A. R., Campos-Sandoval, J. A., et al.

Fig.3.A

Fig.3.A showing Western Blotting in a Chlorocebus aethiops (Grivet monkey) sample from the publication: Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

Differentiation agent PMA increases mitochondrial and nuclear GLS2 protein levels in SH-SY5Y and HepG2 cancer cells. (A) Representative Western blot of nuclear fractions isolated from SH-SY5Y cells after incubation with 1 µM PMA for 12 h and 24 h or dimethyl sulfoxide (DMSO). Blots were probed wi... more

Differentiation agent PMA increases mitochondrial and nuclear GLS2 protein levels in SH-SY5Y and HepG2 cancer cells. (A) Representative Western blot of nuclear fractions isolated from SH-SY5Y cells after incubation with 1 µM PMA for 12 h and 24 h or dimethyl sulfoxide (DMSO). Blots were probed with antibodies against GLS2, p21, p53 and TATA-binding protein (TBP) used as a loading control. Antibodies against COX IV were used to assess cytoplasmic (mitochondrial) contamination. (B) Representative Western blot of nuclear fractions isolated from human GBM T98G cells: wild-type (WT), stably-transfected with vector alone (pcDNA3) and stably-transfected with GLS2 (GAB). Blots were probed with antibodies against GLS2, p53, c-Myc and the loading control TBP. Antibodies against COX IV were used to assess cytoplasmic (mitochondrial) contamination. (C) Representative Western blot of whole cellular extracts isolated from human GBM T98G cells: wild-type (WT) and stably-transfected with GLS2 (GAB). Blots were probed with antibodies against p53 and c-Myc using β-actin as loading control. (D) Confocal microscopy images of GLS2 immunofluorescence in HepG2 cells treated with PMA. Cells grown on cover slips were treated with vehicle DMSO (I–III) or 1 µM PMA in DMSO (II–IV) for 6 h. After incubation, cells were stained with rabbit antibody to GLS2 and Alexa 488 conjugated secondary antibody. Induction of differentiation (II–IV) clearly increased the nuclear (asterisk) and cytoplasmic accumulation of GLS2 protein (arrows) versus untreated cells (I-III). GLS2 also showed some minor nuclear localization without treatment (asterisk in III). Open arrows in IV indicate an intense perinuclear immunostaining of GLS2. Scale bars: 100 μm (I,II) and 10 μm (III, IV). Original Western blots of Fig. 3-A, B and C are shown in Supplemental Fig. S1. Densitometric analysis of the protein bands, normalized to the loading control, are shown on the right of panels A, B and C. For all panels, the values represent the mean (n ≥ 2) and the error bars represent ± SD, except for p21 in A and c-Myc in B (n = 1). Paired Student’s t test was done between PMA treated and untreated cells, and T98G-GAB vs T98G-WT or T98G-pcDNA3.*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. less

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CSB promoter downregulation via histone H3 hypoacetylation is an early determinant of replicative senescence.

In Nat Commun on 6 December 2019 by Crochemore, C., Fernandez-Molina, C., et al.

Fig.7.F

Fig.7.F showing Western Blotting from the publication: CSB promoter downregulation via histone H3 hypoacetylation is an early determinant of replicative senescence.

CSB-dependent senescence is specific to the DDR/p21 pathway. a Enumeration of endogenous γ−H2AX and 53BP1 foci/cell at PNs preceding or during CSB depletion. n = 50–70 cells from three independent experiments; mean ± SEM; one-way ANOVA (γ−H2AX: F = 3.209, DFn = 3, DFd = 220, p = 0.0239; 53BP1: F ... more

CSB-dependent senescence is specific to the DDR/p21 pathway. a Enumeration of endogenous γ−H2AX and 53BP1 foci/cell at PNs preceding or during CSB depletion. n = 50–70 cells from three independent experiments; mean ± SEM; one-way ANOVA (γ−H2AX: F = 3.209, DFn = 3, DFd = 220, p = 0.0239; 53BP1: F = 5.603, DFn = 3, DFd = 220, p = 0.001) with post-hoc Tukey’s test vs. the respective PN16. b Representative confocal acquisitions of irradiated (10 Gy) and non-irradiated (non-IR) IMR-90 immunostained for γ−H2AX (green) and 53BP1 (red), counterstained with Hoechst (blue, nuclei), after maximum intensity projection with the Imaris software; scale bar = 20 µM. Percentage of cells with 0, 1, 2, 3, or >3 (c) γ−H2AX or d 53BP1 foci per nucleus. n = 90 cells from three independent experiments. Mean ± SEM; two-way ANOVA (γ−H2AX: F = 35.77, DFn = 4, DFd = 20, p < 0.001; 53BP1: F = 57.63, DFn = 4, DFd = 20, p < 0.0001) with post-hoc Sidak’s test vs. the respective non-IR. e Quantification of SA-β-gal+ cells of irradiated and non-IR IMR-90 at PN17. n = 180–220 cells/condition from three independent experiments, mean ± SEM; unpaired Student’s t-test (two-tailed) (t = 36.28, DF = 408), p-value vs. non-IR (f) Immunoblots of CSB, HTRA3, p21, and POLG1, HTRA2, p16 (GAPDH F-C staining, loading control, under each blot) in irradiated and non-IR fibroblasts. g Scheme indicating the palbociclib target in the p16 pathway to senescence. h Quantification and i representative images of SA-β-gal+ staining of palbociclib-treated and untreated IMR-90 cells, scale bar = 200 µM. Early-passage fibroblasts (PN20) in normal medium (CTL) or in the presence of 0.2, 0.5, 1, or 5 μM palbociclib for 7 days followed by 1 day of drug withdrawal; n = 800 cells (CTL) and n = 320–380 cells (palbociclib-treated samples) from three independent experiments, mean ± SEM; one-way ANOVA (F = 195.1, DFn = 4, DFd = 2236, p < 0.0001) with post-hoc Tukey’s test. RT-qPCR of jHTRA3, kCSB, and lp21Waf1 and p16Ink4. n = 3 independent experiments, mean ± SD; RM one-way ANOVA (HTRA3: F = 53.07, DF = 4, p < 0.0001; CSB: F = 29.47, DF = 4, p < 0.0001; p21Waf1: F = 13.36, DF = 4, p = 0.0015; p16Ink4: F = 1.587, DF = 4, p = 0.2531) with post-hoc Tukey’s test vs. CTL. m WB of HTRA3, HTRA2, p21, p16, and CSB. Samples on the same blot are framed; each frame displays the respective GAPDH (F-C staining) used as a loading control. Source data are provided as Source Data files. less

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