CiteAb

Fig.3.B showing Western Blotting from the publication: Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

Fig.5.D showing Western Blotting from the publication: Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

Fig.6.C showing Western Blotting from the publication: Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

Fig.5.C showing Western Blotting from the publication: A fast-acting lipid checkpoint in G1 prevents mitotic defects.

Fig.2.A showing Immunohistochemistry in a Mus musculus (House mouse) sample from the publication: p21 facilitates chronic lung inflammation via epithelial and endothelial cells.

Fig.3.H showing Immunohistochemistry-immunofluorescence in a Mus musculus (House mouse) sample from the publication: p21 facilitates chronic lung inflammation via epithelial and endothelial cells.

Fig.1.E showing Western Blotting from the publication: STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells.

Fig.2.K showing Western Blotting from the publication: STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells.

Fig.2.F showing Western Blotting from the publication: STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells.

Fig.3.A showing Western Blotting from the publication: STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells.

Fig.7.A showing Western Blotting from the publication: Centrosome function is critical during terminal erythroid differentiation.

Fig.6.A showing Western Blotting from the publication: Centrosome function is critical during terminal erythroid differentiation.

Fig.2.B showing Western Blotting from the publication: Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Fig.7.G showing Western Blotting from the publication: Cellular senescence limits translational readthrough.

Fig.3.E showing Western Blotting from the publication: CENP-A overexpression promotes distinct fates in human cells, depending on p53 status.

Fig.4.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

Fig.8.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

Fig.8.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

Fig.5.A showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Fig.5.D showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Fig.1.E showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Fig.2.A showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Fig.2.C showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Fig.3.K showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Fig.3.D showing Western Blotting from the publication: HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

88 images found

N-acetylcysteine and raloxifene boost photodynamic therapy against cutaneous squamous cell carcinoma by decreasing TGFβ1 secreted by cancer-associated fibroblasts.

In International Journal of Biological Sciences on 19 May 2025 by Gallego-Rentero, M., Botella, L. M., et al.

Cutaneous squamous cell carcinoma (cSCC) is a highly prevalent skin cancer. While surgery remains the gold standard treatment, non-invasive methods like photodynamic therapy (PDT) stand out for their high efficacy and minimal cosmetic impact. However, resistance to PDT is still a challenge. Numerous cellular processes involved in cancer biology and therapy resistance are regulated by the TGFβ1/SMAD pathway. Using in vitro bidimensional and tridimensional cultures of cSCC cell lines, we studied the development of resistance to PDT in response to TGFβ1 secreted by cancer associated fibroblasts. Our results highlight the TGFβ1 co-receptor endoglin as a key molecular player in the process. Importantly, targeting endoglin expression with N-acetylcysteine (NAC) or raloxifene significantly reduced TGFβ1 levels and effectively prevented resistance. In addition, the combination of PDT with NAC resulted in an improved therapeutic outcome in vivo in SKH-1 mice with cSCC photogenerated by chronic exposition to ultraviolet light. In conclusion, the combination of PDT with NAC or raloxifene enhances PDT efficacy by mitigating resistance mechanisms, which can open new avenues for the treatment of cSCC.
© The author(s).

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Rhus coriaria (Sumac) induces autophagic cell death and inhibits mTOR, p38MAPK and STAT3 pathways in 5fluorouracil-resistant colorectal cancer cells.

In Frontiers in Pharmacology on 3 April 2025 by Nizami, Z. N., Al Azzani, M., et al.

Colorectal cancer is a leading cause of cancer related-death worldwide, and resistance to 5-fluorouracil (5FU, a key component of chemotherapy regimens, is a major clinical concern. We have previously elucidated the effects of Rhus coriaria ethanolic extract (RCE) in triple-negative breast cancer, CRC, and pancreatic cancer cells. Here, we explored the anticancer effects of RCE in parental (HCT-116-WT) and 5FU-resistant HCT-116 (HCT-116-5FU-R) CRC cells.
MTT assay was used to assess cell viability. Muse analyzer was used to assess cell viability, cell cycle distribution, and apoptosis. Additionally, colony formation and growth assays and western blots were performed. In vivo effects of RCE were assessed by an in ovo chick embryo tumor growth assay.
We found that RCE inhibited the viability and colony formation and growth capacities of HCT-116-WT and HCT-116-5FU-R cells. The antiproliferative effects were attributed to DNA damage-mediated impairment of cell cycle at S phase, and induction of Beclin-1-independent autophagy in both cell lines. Mechanistically, inhibition of the mTOR, STAT3 and p38 MAPK pathways was implicated in the latter. Additionally, RCE induced caspase-7-independent apoptosis in HCT-116-WT cells. However, HCT-116-5FU-R cells were resistant to apoptosis through upregulation of survivin, and downregulation of Bax. Using autophagy and proteasome inhibitors, we clarified that autophagy and the proteasome pathway contributed to RCE-mediated cell death in HCT-116-WT and HCT-116-5FU-R cells. Lastly, we confirmed RCE inhibited the growth of both HCT-116-WT and HCT-116-5FU-R xenografts in a chick embryo model.
Collectively, our findings highlight that RCE is a source of phytochemicals that can be used as anticancer agents for 5FU-resistant CRC.
Copyright © 2025 Nizami, Al Azzani, Khaldi, Wali, Magramane, Samad, Eid, Arafat, Al Dhaheri, Attoub and Iratni.

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Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

In ASN NEURO on 18 July 2024 by Guardiola-Diaz, H. M., DiBenedictis, B. T., et al.

The fifteen canonical paracrine fibroblast growth factors (FGFs) are organized in five subfamilies that interact with four FGF-receptors (FGFRs) and heparan sulfate proteoglycan (HSPG) co-receptors. Many of these FGFs are expressed in CNS regions where oligodendrocyte (OL) progenitors originate, migrate or differentiate. FGF2 (basic FGF) is considered a prototype FGF and the information about the effects of FGF signaling on OL-lineage cells has evolved largely from the study of FGF2. However, other FGFs from four subfamilies ((FGF1 (FGF1,-2), FGF4 (FGF4,-5,-6), FGF8 (FGF8,-17,-18) and FGF9 (FGF9,-16,-20)) that can interact with the isoforms of FGFRs expressed in OL-lineage cells may also play important roles. We previously reported OL-responses to FGF8 family members. Here, we investigate the effects of members of the FGF1,-4, and -9 subfamilies on proliferation and differentiation of OL progenitors (OPCs), and on cell cycle re-entry and down-regulation of myelin proteins by mature OLs. We found that while FGF2 induced all these responses strongly, FGF4,-6,-9 could do so only transiently and in the presence of exogenous HSPGs, and that FGF5,-16,-20 could not do so even in the presence of heparin or at higher concentrations. Furthermore, we noted that structurally similar FGFs within subfamilies did not always show similarities in their biological effects on OL-lineage cells. Taken together, these studies reveal that FGFs differ in the way they regulate the OL-lineage cells, emphasizes the selectivity and importance of HSPGs as FGF co-receptors in OL-lineage cells and suggests that structural similarity among FGF-subfamily members may not always predict their overlapping biological functions.

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PRMT5-mediated arginine methylation of FXR1 is essential for RNA binding in cancer cells.

In Nucleic Acids Research on 8 July 2024 by Vijayakumar, A., Majumder, M., et al.

Emerging evidence indicates that arginine methylation promotes the stability of arginine-glycine-rich (RGG) motif-containing RNA-binding proteins (RBPs) and regulates gene expression. Here, we report that post-translational modification of FXR1 enhances the binding with mRNAs and is involved in cancer cell growth and proliferation. Independent point mutations in arginine residues of FXR1's nuclear export signal (R386 and R388) and RGG (R453, R455 and R459) domains prevent it from binding to RNAs that form G-quadruplex (G4) RNA structures. Disruption of G4-RNA structures by lithium chloride failed to bind with FXR1, indicating its preference for G4-RNA structure containing mRNAs. Furthermore, loss-of-function of PRMT5 inhibited FXR1 methylation both in vivo and in vitro, affecting FXR1 protein stability, inhibiting RNA-binding activity and cancer cell growth and proliferation. Finally, the enhanced crosslinking and immunoprecipitation (eCLIP) analyses reveal that FXR1 binds with the G4-enriched mRNA targets such as AHNAK, MAP1B, AHNAK2, HUWE1, DYNC1H1 and UBR4 and controls its mRNA expression in cancer cells. Our findings suggest that PRMT5-mediated FXR1 methylation is required for RNA/G4-RNA binding, which promotes gene expression in cancer cells. Thus, FXR1's structural characteristics and affinity for RNAs preferentially G4 regions provide new insights into the molecular mechanism of FXR1 in oral cancer cells.
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

In Neuron on 3 July 2024 by Lin, L., Zhao, J., et al.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.
Copyright © 2024 Elsevier Inc. All rights reserved.

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Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

In ASN Neuro on 18 July 2024 by Guardiola-Diaz, H. M., DiBenedictis, B. T., et al.

Fig.3.B

Fig.3.B showing Western Blotting from the publication: Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

Effect of different FGFs on oligodendrocyte progenitors in the presence or absence of exogenously added heparin. OPCs were grown in the absence (control, open bars) or presence of heparin (black bars) with FGF1, FGF2, FGF4, FGF5, FGF6, FGF9, FGF16 or FGF20, for 1 d (a), 2 d (b,d) or 3 d (c) and a... more

Effect of different FGFs on oligodendrocyte progenitors in the presence or absence of exogenously added heparin. OPCs were grown in the absence (control, open bars) or presence of heparin (black bars) with FGF1, FGF2, FGF4, FGF5, FGF6, FGF9, FGF16 or FGF20, for 1 d (a), 2 d (b,d) or 3 d (c) and analyzed by immunofluorescence microscopy or immunoblotting for their effects on OPC proliferation and differentiation. A. The quantification of the numbers of BrdU + OPCs, expressed as percentage of total OL-lineage cells (A2B5+/O4+ cells) show that exogenously added heparin significantly increases the proliferation of OPCs by FGF1, FGF2, FGF4, FGF6 and FGF9. b. Quantification of p21cip1 shows significant increase in its level by FGF4 and FGF6 treatment only in the presence of heparin. c. The quantification of the numbers of HPC + OLs, expressed as percentage of total OL-lineage cells (A2B5+/O4+ cells) show that FGF1, FGF2, FGF4, FGF6 and FGF9 significantly decreased their numbers in the presence of exogenously added heparin. d. Quantification of the level of CNP by immunoblotting shows that only FGF4, FGF6 and FGF9 significantly reduces its level in the presence of heparin. Error bars represent SEM; N = 3–7 independent experiments, each performed in triplicate. *p < .05. FGF1, FGF2, FGF4, FGF6, FGF9 were used at 10 ng/ml and FGF5, FGF16, FGF20 at 20 ng/ml. Fresh doses of all FGFs and heparin were re-added on second day with a medium change. Inset, show representative immunoblots for the expression of p21cip1, CNP and actin as protein loading control. less

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A fast-acting lipid checkpoint in G1 prevents mitotic defects.

In Nat Commun on 18 March 2024 by Köberlin, M. S., Fan, Y., et al.

Fig.5.C

Fig.5.C showing Western Blotting from the publication: A fast-acting lipid checkpoint in G1 prevents mitotic defects.

Inhibition of FASN triggers a rapid G1 arrest mediated by increased p21 and reduced Cyclin D.A EGF-released MCF-10A cells (4 h) treated with DMSO or C75. Sample immunofluorescence images for nuclei (Hoechst), p21, and Cyclin D are shown. Scale bar: 10 µm. Data are representative of at least seven... more

Inhibition of FASN triggers a rapid G1 arrest mediated by increased p21 and reduced Cyclin D.A EGF-released MCF-10A cells (4 h) treated with DMSO or C75. Sample immunofluorescence images for nuclei (Hoechst), p21, and Cyclin D are shown. Scale bar: 10 µm. Data are representative of at least seven independent experiments. B Quantification of nuclear protein levels as shown in (A). Data are shown as mean ± SD from at least seven independent experiments, n > 20,000 cells per condition. C EGF-released cells (4 h) treated with indicated inhibitors. Blotted for Cyclin D, p21, and β-actin (loading control). Data are representative of three independent experiments. D Nuclear p21 and Cyclin D protein levels after EGF release and treatment with DMSO or C75 measured by immunofluorescence. Data are representative of at least two independent experiments (data points are means of two technical replicates), n > 17,000 cells per condition. E mRNA levels of CDKN1A (p21) and CCND1 (Cyclin D) measured by qRT-PCR after treatment with DMSO or C75 and normalized to unreleased cells. Data are from at least two independent experiments. F Cyclin D and p21 nuclear protein levels measured by immunofluorescence after 8 h of EGF release in the presence of DMSO or Tunicamycin. Data are from two independent experiments, n > 12,000 cells per condition. B, E, FP values calculated using two-tailed Paired t test. Inhibitor concentrations used unless indicated otherwise: C75 (15 µM), GSK2194069 (50 µM), Tunicamycin (10 µg/ml), Nutlin-3 (10 µM), Torin2 (500 nM). Source data are provided as Source data file. less

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Centrosome function is critical during terminal erythroid differentiation.

In EMBO J on 18 July 2022 by Tátrai, P. & Gergely, F.

Fig.7.A

Fig.7.A showing Western Blotting from the publication: Centrosome function is critical during terminal erythroid differentiation.

Elevated TP53 activity resulting from centrosome or CDK5RAP2 loss is not responsible for abnormal erythroblast differentiationImmunoblot analysis of TP53, P27, and P21 levels in ex vivo cultured Cdk5rap2 wild‐type (WT), heterozygous (HET), and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was... more

Elevated TP53 activity resulting from centrosome or CDK5RAP2 loss is not responsible for abnormal erythroblast differentiationImmunoblot analysis of TP53, P27, and P21 levels in ex vivo cultured Cdk5rap2 wild‐type (WT), heterozygous (HET), and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was used as loading control. ** Indicates unspecific band.Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed.Quantification of cell cycle profiles of ex vivo cultured Trp53HET or Trp53null enucleating EBs with or without CB treatment. EBs were pulsed with BrdU according to Fig 3A. Numbers in brackets represent number of embryos analyzed. See Fig EV6C for cell cycle profiles of TER119pos cells.Quantification of ex vivo cultured Trp53HET or Trp53null enucleating EBs and reticulocytes after 48 h (T48) upon CB treatment. Numbers in brackets represent number of embryos analyzed.Quantification of cell cycle profiles of ex vivo cultured enucleating EBs with the indicated genotypes after 48 h (T48). Number of embryos analyzed is shown in brackets. See Fig EV6F for cell cycle profiles of TER119pos cells.Quantification of ex vivo cultured enucleating EBs and reticulocytes with the indicated genotypes after 48 h (T48). Number of embryos analyzed is shown in brackets.Model shows the origin of EBs enucleating with 2N versus 4N DNA content and the consequences on reticulocyte production.Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). All statistical analysis was based on number of embryos. All statistical significances were determined by one‐way ANOVA with Tukey's multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. less

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Centrosome function is critical during terminal erythroid differentiation.

In EMBO J on 18 July 2022 by Tátrai, P. & Gergely, F.

Fig.6.A

Fig.6.A showing Western Blotting from the publication: Centrosome function is critical during terminal erythroid differentiation.

Terminal erythroid differentiation is impaired in Trp53 and Cdk5rap2 double mutants both ex vivo and in vivoImmunoblot showing levels of TP53, P27, and P21 ex vivo cultured erythroid progenitors upon CB treatment. GAPDH was used as loading control.Quantification of mean protein levels from (A). N... more

Terminal erythroid differentiation is impaired in Trp53 and Cdk5rap2 double mutants both ex vivo and in vivoImmunoblot showing levels of TP53, P27, and P21 ex vivo cultured erythroid progenitors upon CB treatment. GAPDH was used as loading control.Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed.Quantification of cell cycle profiles of ex vivo cultured Trp53HET or Trp53null TER119pos cells following CB treatment from Fig 7C. Numbers in brackets represent number of embryos analyzed.Quantification of number of cell divisions at 48 h (T48) of ex vivo culture following CB treatment. Ex vivo cultured Trp53HET or Trp53null EBs were labeled with PKH26 to measure cell divisions. Number of embryos analyzed is shown in brackets.Quantification of number of cell divisions of CB‐treated enucleating EBs with indicated DNA content from (D). Percentage of enucleating EBs in each category is shown below X‐axis. Number of embryos analyzed is shown in brackets.Quantification of cell cycle profiles of ex vivo cultured TER119pos cells from Fig 7E. Number of embryos analyzed is shown in brackets.Quantification of ex vivo cultured apoptotic (AnnexinVpos) TER119pos cells with indicated genotypes after 48 h (T48). Number of embryos analyzed is shown in brackets.Complete blood count analysis from adult mice with genotypes as indicated. The number of mice analyzed is shown in brackets. RBC = red blood cell. MCV = mean corpuscular volume.Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). Statistical analysis was based on the number of embryos (B‐G) or number of mice (H). All statistical significances were determined by one‐way ANOVA with Tukey's multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. less

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Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

In Cells on 17 April 2022 by Chrysanthou, S., Flores, J. C., et al.

Fig.2.B

Fig.2.B showing Western Blotting from the publication: Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Aberrant upregulation of p21 is responsible for the slow proliferation of Tet1−/− mESCs. (A) Quantification of p21, p27, p15, p16 and p19 mRNA levels in mESCs of indicated genotypes by RT-qPCR. Data normalized to Gapdh expression. Three lines of each genotype were used. (B) Quantification of p21 ... more

Aberrant upregulation of p21 is responsible for the slow proliferation of Tet1−/− mESCs. (A) Quantification of p21, p27, p15, p16 and p19 mRNA levels in mESCs of indicated genotypes by RT-qPCR. Data normalized to Gapdh expression. Three lines of each genotype were used. (B) Quantification of p21 and p53 protein levels in asynchronous (A), G2/M-arrested (0 h) and released (4 h) mESCs of indicated genotypes by Western blot analysis. β-actin was used as a loading control. (C) Quantification of Cdk1, Cdk2 and Cyclin D1 protein levels in asynchronous mESCs of indicated genotypes by Western blot. β-actin was used as a loading control. (D,E) Quantification of p21 mRNA levels by RT-qPCR (D) and protein levels by Western blot (E) in Tet1+/+, Tet1m/m, Tet1−/− mESCs transduced with empty vector (EV) or an shRNA against p21 vector (shp21). (F) Growth curve of mESCs transduced with an empty vector (EV) or an shRNA against p21 vector (shp21) over a 4-day period. (G) The percentage of mESCs of indicated genotypes expressing empty vector (EV) or an shRNA against p21 (shp21) in each phase of the cell cycle 4 h after release from Nocodazole block. (H) Quantification of mRNA levels of p21, Tet1 and pluripotency markers (Oct4 and Nanog) by RT-qPCR in wild type mESCs differentiated (−LIF +RA) for three days. Three independent mESC lines were used. (I) Quantification of mRNA levels of mesoderm markers Bin1 and Eomes by RT-qPCR in Tet1+/+, Tet1m/m and Tet1−/− mESCs expressing empty vector (EV) or an shRNA against p21 (shp21) and cultured in mESC media. Three replicates of each genotype were used. In all panels, data are presented as ± SEM; statistically significant ** p < 0.01 and * p < 0.05 (two-way ANOVA test). less

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Cellular senescence limits translational readthrough.

In Biol Open on 1 December 2021 by Del Toro, N., Lessard, F., et al.

Fig.7.G

Fig.7.G showing Western Blotting from the publication: Cellular senescence limits translational readthrough.

Senescence affects endogenous TR target AGO1x. (A) Images of senescence-associated β galactosidase assay and its quantification in MDA.MB.231 treated with 40 nM camptothecin (therapy-induced senescence (TIS)) or Vehicle (control) for 96 h. Data shows mean of three biological replicates and error ... more

Senescence affects endogenous TR target AGO1x. (A) Images of senescence-associated β galactosidase assay and its quantification in MDA.MB.231 treated with 40 nM camptothecin (therapy-induced senescence (TIS)) or Vehicle (control) for 96 h. Data shows mean of three biological replicates and error bars show SD Students-t-test was performed and P value is indicated. (B) Relative mRNA levels of indicated mRNA in cells as in A. Data shows mean of three biological replicates and error bars show SD Students-t-test was performed and P value is indicated. (C) Immunofluorescence of AGO1x in MDA.MB.231 cells that were treated with 40 nM camptothecin (TIS) or Vehicle (control) or cells that were treated with camptothecin and left to recover for 14 days to allow bypass of senescence (Bypass of TIS). Scale bar=50 µm. A representative image of three biological replicates is shown. D. Quantification of results in (C). A minimum of 50 cells were scored for nuclear staining intensity using Image J. Statistical analysis was performed by ANOVA with Tukey post-test. A.U., arbitrary units. (E) Immunofluorescence of KI67 from MDA.MB.231 cells treated as in C. Scale bar=50 µm. A representative image of three biological replicates is shown. (F) Quantification of the percentage of cells expressing Ki67 shown in E. Statistical analysis was performed by ANOVA with Tukey post-test. (G) Immunoblot against the indicated proteins in MDA.MB.231 cells treated as in C. H3(pS10): phosphorylated Histone H3 at serine 10, and γH2AX: phosphorylated at serine 139 of histone H2AX variant. (H) Regulation of readthrough in senescent cells. less

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CENP-A overexpression promotes distinct fates in human cells, depending on p53 status.

In Commun Biol on 26 March 2021 by Jeffery, D., Gatto, A., et al.

Fig.3.E

Fig.3.E showing Western Blotting from the publication: CENP-A overexpression promotes distinct fates in human cells, depending on p53 status.

Sensitivity to X-irradiation upon CENP-A overexpression is dependent on functional p53.a Scheme of colony formation assays (CFAs) and change of p53 status with relative CENP-A protein levels over time for TetOn-CENPA-FLAG-HA cell lines. WT p53 is transiently activated in HeLa cells by a short hea... more

Sensitivity to X-irradiation upon CENP-A overexpression is dependent on functional p53.a Scheme of colony formation assays (CFAs) and change of p53 status with relative CENP-A protein levels over time for TetOn-CENPA-FLAG-HA cell lines. WT p53 is transiently activated in HeLa cells by a short heat shock, while p53 is stably inactivated in MCF10-2A cells by expression of a p53 dominant-negative peptide (p53-DN). b Temporary change of p53 status in HeLa cells by 1 h heat shock (HS). Western blot of HeLa cell TCEs at indicated time points following 1 h at 42 or 37 °C control. Temporary increase of p53 and subsequently p21. 1x load = ~33,000 cells. Primary antibodies are indicated on the right. # = high sensitivity ECL exposure. H4 used as loading control. c Increase of p53 levels in HeLa cells by heat shock. Western blot of TCEs after 1 h at 42 or 37 °C control. Loaded at 8x, 4x, 2x, 1x concentrations, where 1x load = ~12,500 cells. Primary antibodies are indicated on the right. # = high sensitivity ECL exposure. H4 used as loading control. Representative western blot from three independent experiments is shown. d HeLa S3 CFAs pertaining to scheme in (a). Each dot represents a single biological replicate. Plots show mean and 95% confidence interval. Statistical significance tested by two-tailed Welch’s t test with Bonferroni cutoff at a p value of 0.0125 (α = 0.05). * = p value < 0.01. e Change of p53 status in MCF10-2A TetOn-CENPA-FLAG-HA cells. Western blot of TCEs after 24 h of 10 µM Nutlin-3 treatment or DMSO control, as indicated. Dominant negative p53 peptide stabilizes p53 and suppresses its activation of p21. 1x load corresponds to total protein extract from ~30,000 cells. Primary antibodies are indicated on the right. # = high sensitivity ECL exposure. H4 used as loading control. Representative western blot from three independent experiments is shown. f MCF10-2A CFAs pertaining to scheme in (a). Each dot represents a single biological replicate. Plots show mean and 95% confidence interval. Statistical significance tested by two-tailed Welch’s t test with Bonferroni cutoff at a p value of 0.0125 (α = 0.05). *** = p value < 0.0001. See also Supplementary Fig. 1C, D for CFAs following range of Dox and X-irradiation doses and Supplementary Fig. 1E for representative CFA images. less

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IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

In PLoS Genet on 1 March 2021 by Lemarié, M., Bottardi, S., et al.

Fig.8.D

Fig.8.D showing Western Blotting in a Mus musculus (House mouse) sample from the publication: IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

IKAROS is required to prevent the overexpression of P21WAF1/CIP1 upon DNA damage induction.Western blot analyses of IKAROS, TP53, P21WAF1/CIP1 and GAPDH or ACTIN (loading controls) expression in total cell lysates. All experiments were performed in triplicate and representative results are shown ... more

IKAROS is required to prevent the overexpression of P21WAF1/CIP1 upon DNA damage induction.Western blot analyses of IKAROS, TP53, P21WAF1/CIP1 and GAPDH or ACTIN (loading controls) expression in total cell lysates. All experiments were performed in triplicate and representative results are shown in panels A-D. (A) IkWT or IkNull Ter119+ cells were harvested after 48h of co-culture with OP9 or OP9-DL1 cells, irradiated (3.5 Gy) and collected 3h post-irradiation. (B) Protein expression; the relative expression levels of IKAROS in G1E-ER4 cells stably transfected with either scrCTL (non-target control) or shIK (IKAROS shRNA), were obtained by dividing the densitometric values (calculated using the Multi Gauge V3.0 software; right panel) of IKAROS bands by the values of ACTIN bands (IK/ACTIN ratios); y axis: IKAROS/ACTIN protein level in shIK relative to scrCTL. (C) G1E-ER4 cells transfected either with scrCTL (non-target control) or shIK (IKAROS shRNA); stable clones were transiently transfected with either siNT (control) or siTP53 and irradiated (3.5 Gy); cells were collected 3h post-irradiation. (D) G1E-ER4 or erythroid cells were treated with 5 μM or 10 μM lenalidomide; cells were collected 24h after the treatment; the relative expression levels were obtained dividing the densitometric values (calculated using the Multi Gauge V3.0 software) of IKAROS bands by the values of GAPDH bands (IK/GAPDH ratios) or P21WAF1/CIP1 bands by the value of GAPDH bands (P21WAF1/CIP1/GAPDH ratios); relative expression levels are represented by bars and plotted as the mean ± Standard Deviation of the measurements (right panel); *: p ≤ 0.05 by Student’s t-test; IK1: IKAROS splicing isoform 1; IK2: IKAROS splicing isoform 2. less

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IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

In PLoS Genet on 1 March 2021 by Lemarié, M., Bottardi, S., et al.

Fig.8.C

Fig.8.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: IKAROS is required for the measured response of NOTCH target genes upon external NOTCH signaling.

IKAROS is required to prevent the overexpression of P21WAF1/CIP1 upon DNA damage induction.Western blot analyses of IKAROS, TP53, P21WAF1/CIP1 and GAPDH or ACTIN (loading controls) expression in total cell lysates. All experiments were performed in triplicate and representative results are shown ... more

IKAROS is required to prevent the overexpression of P21WAF1/CIP1 upon DNA damage induction.Western blot analyses of IKAROS, TP53, P21WAF1/CIP1 and GAPDH or ACTIN (loading controls) expression in total cell lysates. All experiments were performed in triplicate and representative results are shown in panels A-D. (A) IkWT or IkNull Ter119+ cells were harvested after 48h of co-culture with OP9 or OP9-DL1 cells, irradiated (3.5 Gy) and collected 3h post-irradiation. (B) Protein expression; the relative expression levels of IKAROS in G1E-ER4 cells stably transfected with either scrCTL (non-target control) or shIK (IKAROS shRNA), were obtained by dividing the densitometric values (calculated using the Multi Gauge V3.0 software; right panel) of IKAROS bands by the values of ACTIN bands (IK/ACTIN ratios); y axis: IKAROS/ACTIN protein level in shIK relative to scrCTL. (C) G1E-ER4 cells transfected either with scrCTL (non-target control) or shIK (IKAROS shRNA); stable clones were transiently transfected with either siNT (control) or siTP53 and irradiated (3.5 Gy); cells were collected 3h post-irradiation. (D) G1E-ER4 or erythroid cells were treated with 5 μM or 10 μM lenalidomide; cells were collected 24h after the treatment; the relative expression levels were obtained dividing the densitometric values (calculated using the Multi Gauge V3.0 software) of IKAROS bands by the values of GAPDH bands (IK/GAPDH ratios) or P21WAF1/CIP1 bands by the value of GAPDH bands (P21WAF1/CIP1/GAPDH ratios); relative expression levels are represented by bars and plotted as the mean ± Standard Deviation of the measurements (right panel); *: p ≤ 0.05 by Student’s t-test; IK1: IKAROS splicing isoform 1; IK2: IKAROS splicing isoform 2. less

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