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Fig.2.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

Fig.2.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

Fig.2.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

Fig.2.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

Fig.1.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

Fig.3.C showing Western Blotting from the publication: KLF4α stimulates breast cancer cell proliferation by acting as a KLF4 antagonist.

Fig.2.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane.

Fig.2.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane.

Fig.5.B showing Western Blotting from the publication: Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

10 images found

Miro1 expression alters global gene expression, ERK1/2 phosphorylation, oxidation, and cell cycle progression

Preprint on BioRxiv : the Preprint Server for Biology on 6 November 2024 by Shannon, N., Raymond, C., et al.

Subcellular mitochondrial positioning in cells is necessary for localized energy and signaling requirements. Mitochondria are strategically trafficked throughout the cytoplasm via the actin cytoskeleton, microtubule motor proteins, and adaptor proteins. Miro1, an outer mitochondrial membrane adaptor protein, is necessary for attachment of mitochondria to microtubule motor proteins for trafficking. Previous work showed when Miro1 is deleted (Miro1 -/- ) from mouse embryonic fibroblasts (MEFs), the mitochondria become sequestered to the perinuclear space, disrupting subcellular energy and reactive oxygen species gradients. Here, we show that Miro1 -/- MEFs grow slower compared to Miro1 +/+ and Miro1 -/- MEFs stably re-expressing the Myc-Miro1 plasmid. Miro1 -/- MEFs have a have a cell cycle defect with decreased percentage of cells in G1 and increased cells in the S phase of the cell cycle. We conducted the first ever RNA sequencing experiment dependent upon Miro1 expression and found differential expression in cell proliferation and migration genes upon deletion of Miro1, including the MAP Kinase signaling pathway. We find that ERK1/2 phosphorylation is elevated both spatially (cytoplasm and nucleus) and temporally following serum stimulation in Miro1 -/- MEFs. We investigated the expression levels and oxidation of the Dual Specificity Phosphatases (DUSP1-6), ERK1/2 target phosphatases. We found no differences in DUSP1-6 expression and oxidation under asynchronous and synchronized cells. Lastly, we evaluated the oxidation status of ERK1/2 and found an increase in ERK1/2 oxidation in the Miro1 -/- MEFs compared to Miro1 +/+ and Myc-Miro1. These data highlight transcriptional control based off Miro1 expression and demonstrate the highly dynamic regulation of ERK1/2 upon deletion of Miro1 that may support the observed cell cycle and proliferation defects.

Putting a Kink in HIV-1 Particle Infectivity: Rocaglamide Inhibits HIV-1 Replication by Altering Gag-Genomic RNA Interaction.

In Viruses on 23 September 2024 by Rosenfeld, P., Singh, G., et al.

Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA), a known modulator of eIF4A1 function, as an inhibitor of HIV-1 replication in primary CD4+ T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather, the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA, but together, they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together, several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions.

Blastocoel expansion and AMOT degradation cooperatively promote YAP nuclear localization during epiblast formation

Preprint on BioRxiv : the Preprint Server for Biology on 18 August 2024 by Maeda, H. & Sasaki, H.

The epiblast is a pluripotent cell population formed in the late blastocyst stage of preimplantation embryos. During the process of epiblast formation from the inner cell mass (ICM) of the early blastocyst, activation of the Hippo pathway transcription factor TEAD by the nuclear translocation of the coactivator protein YAP is required for the robust expression of pluripotency factors. However, the mechanisms that alter YAP localization during epiblast formation remain unknown. Here, we reveal two such mechanisms. Expansion of the blastocoel promotes nuclear YAP localization by increasing cytoplasmic F-actin and reducing YAP phosphorylation. Additionally, cell differentiation regulates YAP. Expression of the junctional Hippo component, AMOT, gradually decreases during epiblast formation through a tankyrase-mediated degradation. SOX2 expression in the ICM is necessary for the reduction of AMOT and YAP phosphorylation. These two mechanisms function in parallel. Thus, the blastocoel-F-actin and SOX2-AMOT axes cooperatively suppress YAP phosphorylation and promote YAP nuclear localization during epiblast formation. The cooperation of these two distinct mechanisms likely contributes to the robustness of epiblast cell differentiation.

Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.

In Life Science Alliance on 1 September 2023 by Kim, J. Y., Atanassov, I., et al.

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.
© 2023 Kim et al.

Overexpressed Nup88 stabilized through interaction with Nup62 promotes NF-κB dependent pathways in cancer.

In Frontiers in Oncology on 28 February 2023 by Singh, U., Bindra, D., et al.

Bidirectional nucleo-cytoplasmic transport, regulating several vital cellular processes, is mediated by the Nuclear Pore Complex (NPC) comprising the nucleoporin (Nup) proteins. Nup88, a constituent nucleoporin, is overexpressed in many cancers, and a positive correlation exists between progressive stages of cancer and Nup88 levels. While a significant link of Nup88 overexpression in head and neck cancer exists but mechanistic details of Nup88 roles in tumorigenesis are sparse. Here, we report that Nup88 and Nup62 levels are significantly elevated in head and neck cancer patient samples and cell lines. We demonstrate that the elevated levels of Nup88 or Nup62 impart proliferation and migration advantages to cells. Interestingly, Nup88-Nup62 engage in a strong interaction independent of Nup-glycosylation status and cell-cycle stages. We report that the interaction with Nup62 stabilizes Nup88 by inhibiting the proteasome-mediated degradation of overexpressed Nup88. Overexpressed Nup88 stabilized by interaction with Nup62 can interact with NF-κB (p65) and sequesters p65 partly into nucleus of unstimulated cells. NF-κB targets like Akt, c-myc, IL-6 and BIRC3 promoting proliferation and growth are induced under Nup88 overexpression conditions. In conclusion, our data indicates that simultaneous overexpression of Nup62 and Nup88 in head and neck cancer stabilizes Nup88. Stabilized Nup88 interacts and activates p65 pathway, which perhaps is the underlying mechanism in Nup88 overexpressing tumors.
Copyright © 2023 Singh, Bindra, Samaiya and Mishra.

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IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

In Discov Oncol on 11 May 2021 by Mills, J. V., Osher, E., et al.

Fig.1.B

Full length IGF-1Rα- and β-subunits are detectable in the nucleus of prostate cancer cells a Domain structure of mature IGF-1R at the plasma membrane, showing extracellular α-subunit (green), transmembrane β-subunit (red), extracellular (ED), transmembrane (TM), kinase domains (KD) and disulphide bonds, modified from [3]. b, c Western blots showing detection of both IGF-1Rα- and β-subunits in the nucleus of DU145 (b) and 22Rv1 (c) cells. Cells were serum-starved for 24 h, treated with solvent (4.5 µM HCl) or IGF-1 (50 nM) for 20 min before fractionation. All western blots shown are representative of 3 independent biological repeats. Arrowheads: 220 kDa IGF-1R proreceptor. d Images showing immunofluorescence staining of IGF-1Rα and IGF-1Rβ in DU145 cells. DU145 cells were serum-starved and IGF-treated as in b-c followed by immunofluorescent staining. Graphs: mean ± SD nuclear IGF-1Rα- and β-subunits (n ≥ 30 cells per condition for n = 3 biological repeats). Data were analysed by unpaired student’s t test and quantification of nuclear levels of IGF-1Rα- and β-subunits showed a significant increase on treatment with IGF-1 (*p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001) e Images showing IGF-1Rα and IGF-1Rβ immunofluorescence in DU145 cells. Cells were grown in full medium with 10% FCS and incubated with solvent (0.1% DMSO) or IGF-1R inhibitor BMS-754807 (100 nM) for 6 h at 37 °C and processed for immunofluorescence staining. Graphs: mean ± SD nuclear IGF-1Rα- and β-subunits (n ≥ 30 cells per condition for n = 2 biological repeats). Data were analysed by unpaired student’s t test and quantification of nuclear levels of IGF-1Rα- and β-subunits showed a significant decrease on treatment with BMS-754807 (*p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001) f Images showing cultured DU145 cells stained with Alexa-488 secondary antibodies as used in d and e in the absence of primary antibody. These images represent a negative control for immunofluorescence staining and demonstrate the specificity of the IGF-1Rα- and β-subunit signals shown in d and e less

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IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits.

In Discov Oncol on 11 May 2021 by Mills, J. V., Osher, E., et al.

Fig.1.C

Collected and cropped from Discov Oncol by CiteAb, provided under a CC-BY license

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KLF4α stimulates breast cancer cell proliferation by acting as a KLF4 antagonist.

In Oncotarget on 19 July 2016 by Ferralli, J., Chiquet-Ehrismann, R., et al.

Fig.3.C

Characterization of KLF4α expression(A) MDA-MB-231 cells were transfected with the indicated plasmids and immunoblotted with α-KLF4, which only detects KLF4(FL), with α-myc, which detects both myc-tagged variants of KLF4, and with α-GN330, which specifically recognizes KLF4α. *background band. (B) Immunofluorescent stainings of transfected MDA-MB-231 cells 24 h post-transfection show nuclear staining for KLF4(FL) and cytoplasmic staining for KLF4α. Hoechst stain was used for the visualization of nuclei. Scale bar: 50 μm (top); 10 μm (close-up). The bottom row indicates the secondary antibody only controls merged with the respective Hoechst staining (left side: goat-anti-rabbit IgG-FITC; right side: goat-anti-mouse IgG-FITC). (C) Transfected MDA-MB-231 cells were extracted for the cytoplasmic and the nuclear fractions 36 hours after transfection and immunoblotted. KLF4α is only found in the cytoplasmic, GAPDH-positive fraction, whereas most of KLF4(FL) is present in the nuclear, LaminA/C-positive fraction. * background band. (D) Western blot analysis of protein extracts of the normal breast cell line (MCF10A), four breast cancer cell lines as well as a p53-mutant Li Fraumeni cell line (172) in comparison to KLF4α-transfected MDA-MB-231 cells for KLF4α levels. Note that endogenous KLF4α (∼18kDa) is detectable in MDA-MB-175, MDA-MB-231, and 172 cells. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. less

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Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane.

In Nucleic Acids Res on 31 March 2015 by Jeong, K., Kwon, H., et al.

Fig.2.A

pY19-Cav-2 association with lamin A/C is essential for the sustenance of Tyr-19 phosphorylation from the dephosphorylation by nuclear PTP1B. (A and B) Lamin A/C association of Cav-2 in the INM for the maintenance of Tyr-19 phosphorylation. Scramble control or siLamin A/C-transfected Hirc-B cells (A) or Cav-2-GFP- or Δ47–86-Cav-2-GFP-expressed HEK293T cells (B) treated with or without 100 nM insulin for 30 min were subjected to nuclear fractionation. Equal amounts of protein for each fraction were analyzed by immunoblotting (n = 3). E-cadherin, α-tubulin, and lamin A/C and emerin were detected as markers for membrane (M), cytoplasmic (C) and nuclear (N) fractions, respectively. (C and D) Nuclear dephosphorylation of pY19-Cav-2 defective in its association with lamin A/C. Nuclear lysates from scramble control or siLamin A/C-transfected Hirc-B cells (C) or Cav-2-GFP- or Δ47–86-Cav-2-GFP-expressed HEK293T cells (D) treated with or without 200 μM vanadate for 30 min followed by incubation with or without 100 nM insulin for 30 min were subjected to immunoblotting (n = 3). (E) Dephosphorylation of pY19-Cav-2 defective in the lamin A/C association by nuclear PTP1B. Nuclear lysates from Cav-2-GFP or Δ47–86-Cav-2-GFP and scramble control or siPTP1B-co-transfected HEK293T cells treated with or without 100 nM insulin for 30 min were subjected to immunoblotting (n = 3). (F and G) Interaction of the lamin A/C association-defective Cav-2 with nuclear PTP1B. Nuclear lysates from scramble control or siLamin A/C-transfected Hirc-B cells (F) or Cav-2-GFP- or Δ47–86-Cav-2-GFP-expressed HEK293T cells (G) treated with or without 100 nM insulin for 30 min were immunoprecipitated with anti-Cav-2 (F) or anti-GFP (G) antibody and subjected to immunoblotting (n = 3). less

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Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane.

In Nucleic Acids Res on 31 March 2015 by Jeong, K., Kwon, H., et al.