antibodies against cyclin c  (Cell Signaling Technology Inc)

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: Pharmacological inhibition of CDK8/19 alleviates PRCC-TFE3 induced oncogene-induced senescence (OIS) . (A) Growth curves of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells treated with the CDK8/19 inhibitor MSC2530818 (100 nM) in the presence or absence of Dox. Cell numbers were measured at the indicated time points (n = 3). MSC2530818 reduced basal proliferation in Dox(–) cells; however, it markedly alleviated the growth suppression caused by PRCC-TFE3 induction upon Dox treatment. (B) Senescence-associated β-galactosidase (SA-β-gal) staining of PRCC-TFE3 Dox inducible HK-2 cells cultured for 5 days in the presence or absence of the CDK8/19 inhibitor MSC2530818. PRCC-TFE3 induction robustly increased SA-β-gal positive senescent cells, whereas MSC2530818 treatment markedly attenuated PRCC-TFE3 induced cellular senescence. Black arrows indicate SA-β-gal positive cells. Representative images from three independent experiments are shown. Scale bar, 200 μm. (C) RT-qPCR analysis of lamin B1 mRNA levels in non-induced and PRCC-TFE3 induced HK-2 cells treated with MSC2530818 (100 nM) (n = 3). MSC2530818 treatment reduced basal lamin B1 expression in non-induced cells; however, no additional decrease in lamin B1 levels was observed upon PRCC-TFE3 induction in the presence of MSC2530818, indicating that CDK8/19 inhibition prevents PRCC-TFE3 associated lamin B1 downregulation. (D) RT-qPCR analysis of senescence-associated secretory phenotype (SASP) factor mRNAs (IL1A, IL6, and VEGF) in non-induced and PRCC-TFE3 induced HK-2 cells treated with MSC2530818 (100 nM) (n = 3). PRCC-TFE3 induction was associated with robust upregulation of SASP factor expression, consistent with the induction of oncogene-induced senescence. Pharmacological inhibition of CDK8/19 by MSC2530818 markedly attenuated this SASP response, indicating suppression of PRCC-TFE3 induced senescence. (E) Cell-cycle analysis of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells cultured for 3 days under the indicated combinations of Dox (−/+) and the CDK8/19 inhibitor MSC2530818 (100 nM). Cells were labeled with BrdU for 90 min, fixed, stained with phycoerythrin (PE)-conjugated anti-BrdU antibodies, and counterstained with propidium iodide (PI). Representative flow cytometry plots are shown (top), and quantitative analyses of the G0/G1, S, and G2/M populations are summarized (bottom) (n = 3). MSC2530818 treatment largely abrogated PRCC-TFE3 induced cell cycle arrest, restoring S phase entry. (F) Immunofluorescence staining of Cyclin C and HA-tagged PRCC-TFE3 in PRCC-TFE3 Dox-inducible HK-2 cells cultured in the absence (left) or presence of doxycycline (right). Upon PRCC-TFE3 induction, Cyclin C exhibits prominent punctate nuclear localization. Nuclei were counterstained with DAPI. Representative images are shown. Scale bars, 10 μm. Quantification of cells displaying Cyclin C nuclear puncta is shown on the right (n = 3). (G) Chromatin immunoprecipitation (ChIP)-qPCR analysis of HA-tagged PRCC-TFE3 and Cyclin C occupancy at the indicated gene regulatory regions in PRCC-TFE3 Dox-inducible HK-2 cells (n = 3). Induction of PRCC-TFE3 resulted in robust recruitment of PRCC-TFE3 to these genomic regions, accompanied by a concomitant increase in Cyclin C binding at the same loci, suggesting coordinated engagement of PRCC-TFE3 and Cyclin C at shared transcriptional regulatory sites. Data are presented as means ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: The cells were then incubated overnight at 4 °C with primary antibodies against Cyclin C (68179; Cell Signaling Technology) and HA (3F10; Roche) diluted in 0.1% TBST.

Techniques: Inhibition, Staining, Cell Culture, Quantitative RT-PCR, Expressing, Cell Cycle Assay, Labeling, Flow Cytometry, Immunofluorescence, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: The Mediator kinase module is required for the transcriptional and oncogenic functions of chimeric TFE3 . (A) RT-qPCR analysis of representative TFE3 target genes (GPNMB, FNIP2, and RRAGD) in patient derived TFE3-RCC cell line harboring endogenous PRCC-TFE3 (UOK124), treated with or without the CDK8/19 inhibitor MSC2530818 (100 nM) (n = 3). CDK8/19 inhibition attenuated the expression of TFE3 target genes in both TFE3-RCC cell lines. (B) Flow cytometric analysis of lysosomal content in UOK124 cells treated with the CDK8/19 inhibitor MSC2530818 (100 nM) or transduced with shRNA targeting TFE3 (shTFE3). Cells were stained with LysoPrime Green to assess lysosomal volume. Representative flow cytometry histograms (left) and quantitative analysis of mean fluorescence intensity (right) are shown (n = 3). Both MSC2530818 treatment and TFE3 knockdown significantly reduced lysosomal content, indicating suppression of PRCC-TFE3 dependent lysosomal activation. (C) Colony formation assay of UOK124 cells treated with DMSO (Control) or MSC2530818 (100 nM) (n=3). Representative images of colonies are shown (left). Quantification demonstrates that MSC2530818 significantly suppresses anchorage independent colony formation by reducing colony number, without a significant effect on colony size (right). (D) In vitro cell proliferation analysis following genetic ablation of CCNC in UOK124 cells. Successful knockout of CCNC was confirmed by western blotting, with β-actin shown as a loading control (top). Cell proliferation was assessed by cell counting over time (n=8), and growth curves are shown for control and CCNC KO cells (bottom). Genetic ablation of CCNC significantly suppressed cell proliferation compared with control cells. (E) In vivo tumorigenicity of CCNC KO UOK124 cells in nude mice. Control or CCNC KO UOK124 cells (1 × 10 6 cells per injection) were subcutaneously injected into the left and right flanks of the same nude mice, respectively (n = 6 mice). Tumor volumes were measured at the indicated time points and are shown in the upper left panel. Representative images of tumors excised at day 39 after injection are shown in the upper right panel. Histological and immunohistochemical analyses of tumors derived from control and CCNC KO UOK124 cells. Representative hematoxylin and eosin (H&E) staining is shown in the left panels, and Cyclin C immunohistochemical staining is shown in the right panels. Scale bars, 50 μm. Tumor weights at the endpoint are summarized in the lower right panel. Genetic ablation of CCNC dramatically suppressed tumor growth and reduced final tumor weight compared with control tumors. (F) Tumor growth in an orthotopic syngeneic TFE3 RCC model derived from PRCC-TFE3 knock in mice treated with vehicle or the CDK8 and CDK19 inhibitor SEL120. Tumors derived from PRCC-TFE3 knock-in mice were orthotopically implanted into syngeneic recipient mice, which were subsequently treated with vehicle or SEL120 (60 mg/kg, oral administration, 5 days per week, n=10/arm) for 8 weeks. Tumor growth was monitored longitudinally by ultrasound imaging, and tumor volumes were quantified at the indicated time points. SEL120 treatment significantly suppressed tumor growth compared with vehicle control. Data shown are representative of two independent experiments. (G) Proposed schematic model summarizing the central findings of this study. Proposed model illustrating the interaction between TFE3 fusion proteins and the Mediator complex via the Cyclin C-CDK8/19 kinase module. While enhancer-promoter communication and chromatin organization are not directly assessed in this study, the model reflects established functions of Mediator in transcriptional regulation. Disruption of this axis, through genetic ablation or pharmacological inhibition of CDK8/19, impairs TFE3 fusion dependent transcription and suppresses tumor growth in cellular and in vivo models, identifying the Mediator kinase module as a potential therapeutic target in TFE3 RCC. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against a control group, or two-way ANOVA followed by Sidak’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: The cells were then incubated overnight at 4 °C with primary antibodies against Cyclin C (68179; Cell Signaling Technology) and HA (3F10; Roche) diluted in 0.1% TBST.

Techniques: Quantitative RT-PCR, Derivative Assay, Inhibition, Expressing, Transduction, shRNA, Staining, Flow Cytometry, Fluorescence, Knockdown, Activation Assay, Colony Assay, Control, In Vitro, Knock-Out, Western Blot, Cell Counting, In Vivo, Injection, Immunohistochemical staining, Knock-In, Imaging, Disruption, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: Pharmacological inhibition of CDK8/19 alleviates PRCC-TFE3 induced oncogene-induced senescence (OIS) . (A) Growth curves of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells treated with the CDK8/19 inhibitor MSC2530818 (100 nM) in the presence or absence of Dox. Cell numbers were measured at the indicated time points (n = 3). MSC2530818 reduced basal proliferation in Dox(–) cells; however, it markedly alleviated the growth suppression caused by PRCC-TFE3 induction upon Dox treatment. (B) Senescence-associated β-galactosidase (SA-β-gal) staining of PRCC-TFE3 Dox inducible HK-2 cells cultured for 5 days in the presence or absence of the CDK8/19 inhibitor MSC2530818. PRCC-TFE3 induction robustly increased SA-β-gal positive senescent cells, whereas MSC2530818 treatment markedly attenuated PRCC-TFE3 induced cellular senescence. Black arrows indicate SA-β-gal positive cells. Representative images from three independent experiments are shown. Scale bar, 200 μm. (C) RT-qPCR analysis of lamin B1 mRNA levels in non-induced and PRCC-TFE3 induced HK-2 cells treated with MSC2530818 (100 nM) (n = 3). MSC2530818 treatment reduced basal lamin B1 expression in non-induced cells; however, no additional decrease in lamin B1 levels was observed upon PRCC-TFE3 induction in the presence of MSC2530818, indicating that CDK8/19 inhibition prevents PRCC-TFE3 associated lamin B1 downregulation. (D) RT-qPCR analysis of senescence-associated secretory phenotype (SASP) factor mRNAs (IL1A, IL6, and VEGF) in non-induced and PRCC-TFE3 induced HK-2 cells treated with MSC2530818 (100 nM) (n = 3). PRCC-TFE3 induction was associated with robust upregulation of SASP factor expression, consistent with the induction of oncogene-induced senescence. Pharmacological inhibition of CDK8/19 by MSC2530818 markedly attenuated this SASP response, indicating suppression of PRCC-TFE3 induced senescence. (E) Cell-cycle analysis of PRCC-TFE3 doxycycline (Dox)-inducible HK-2 cells cultured for 3 days under the indicated combinations of Dox (−/+) and the CDK8/19 inhibitor MSC2530818 (100 nM). Cells were labeled with BrdU for 90 min, fixed, stained with phycoerythrin (PE)-conjugated anti-BrdU antibodies, and counterstained with propidium iodide (PI). Representative flow cytometry plots are shown (top), and quantitative analyses of the G0/G1, S, and G2/M populations are summarized (bottom) (n = 3). MSC2530818 treatment largely abrogated PRCC-TFE3 induced cell cycle arrest, restoring S phase entry. (F) Immunofluorescence staining of Cyclin C and HA-tagged PRCC-TFE3 in PRCC-TFE3 Dox-inducible HK-2 cells cultured in the absence (left) or presence of doxycycline (right). Upon PRCC-TFE3 induction, Cyclin C exhibits prominent punctate nuclear localization. Nuclei were counterstained with DAPI. Representative images are shown. Scale bars, 10 μm. Quantification of cells displaying Cyclin C nuclear puncta is shown on the right (n = 3). (G) Chromatin immunoprecipitation (ChIP)-qPCR analysis of HA-tagged PRCC-TFE3 and Cyclin C occupancy at the indicated gene regulatory regions in PRCC-TFE3 Dox-inducible HK-2 cells (n = 3). Induction of PRCC-TFE3 resulted in robust recruitment of PRCC-TFE3 to these genomic regions, accompanied by a concomitant increase in Cyclin C binding at the same loci, suggesting coordinated engagement of PRCC-TFE3 and Cyclin C at shared transcriptional regulatory sites. Data are presented as means ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: The following primary antibody was used: rabbit monoclonal anti Cyclin C (#68179; CST; 1:200).

Techniques: Inhibition, Staining, Cell Culture, Quantitative RT-PCR, Expressing, Cell Cycle Assay, Labeling, Flow Cytometry, Immunofluorescence, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: Neoplasia (New York, N.Y.)

Article Title: The Cyclin C-CDK8/19 Mediator kinase module controls PRCC-TFE3 driven senescence in renal epithelium and tumorigenesis in TFE3-RCC

doi: 10.1016/j.neo.2026.101296

Figure Lengend Snippet: The Mediator kinase module is required for the transcriptional and oncogenic functions of chimeric TFE3 . (A) RT-qPCR analysis of representative TFE3 target genes (GPNMB, FNIP2, and RRAGD) in patient derived TFE3-RCC cell line harboring endogenous PRCC-TFE3 (UOK124), treated with or without the CDK8/19 inhibitor MSC2530818 (100 nM) (n = 3). CDK8/19 inhibition attenuated the expression of TFE3 target genes in both TFE3-RCC cell lines. (B) Flow cytometric analysis of lysosomal content in UOK124 cells treated with the CDK8/19 inhibitor MSC2530818 (100 nM) or transduced with shRNA targeting TFE3 (shTFE3). Cells were stained with LysoPrime Green to assess lysosomal volume. Representative flow cytometry histograms (left) and quantitative analysis of mean fluorescence intensity (right) are shown (n = 3). Both MSC2530818 treatment and TFE3 knockdown significantly reduced lysosomal content, indicating suppression of PRCC-TFE3 dependent lysosomal activation. (C) Colony formation assay of UOK124 cells treated with DMSO (Control) or MSC2530818 (100 nM) (n=3). Representative images of colonies are shown (left). Quantification demonstrates that MSC2530818 significantly suppresses anchorage independent colony formation by reducing colony number, without a significant effect on colony size (right). (D) In vitro cell proliferation analysis following genetic ablation of CCNC in UOK124 cells. Successful knockout of CCNC was confirmed by western blotting, with β-actin shown as a loading control (top). Cell proliferation was assessed by cell counting over time (n=8), and growth curves are shown for control and CCNC KO cells (bottom). Genetic ablation of CCNC significantly suppressed cell proliferation compared with control cells. (E) In vivo tumorigenicity of CCNC KO UOK124 cells in nude mice. Control or CCNC KO UOK124 cells (1 × 10 6 cells per injection) were subcutaneously injected into the left and right flanks of the same nude mice, respectively (n = 6 mice). Tumor volumes were measured at the indicated time points and are shown in the upper left panel. Representative images of tumors excised at day 39 after injection are shown in the upper right panel. Histological and immunohistochemical analyses of tumors derived from control and CCNC KO UOK124 cells. Representative hematoxylin and eosin (H&E) staining is shown in the left panels, and Cyclin C immunohistochemical staining is shown in the right panels. Scale bars, 50 μm. Tumor weights at the endpoint are summarized in the lower right panel. Genetic ablation of CCNC dramatically suppressed tumor growth and reduced final tumor weight compared with control tumors. (F) Tumor growth in an orthotopic syngeneic TFE3 RCC model derived from PRCC-TFE3 knock in mice treated with vehicle or the CDK8 and CDK19 inhibitor SEL120. Tumors derived from PRCC-TFE3 knock-in mice were orthotopically implanted into syngeneic recipient mice, which were subsequently treated with vehicle or SEL120 (60 mg/kg, oral administration, 5 days per week, n=10/arm) for 8 weeks. Tumor growth was monitored longitudinally by ultrasound imaging, and tumor volumes were quantified at the indicated time points. SEL120 treatment significantly suppressed tumor growth compared with vehicle control. Data shown are representative of two independent experiments. (G) Proposed schematic model summarizing the central findings of this study. Proposed model illustrating the interaction between TFE3 fusion proteins and the Mediator complex via the Cyclin C-CDK8/19 kinase module. While enhancer-promoter communication and chromatin organization are not directly assessed in this study, the model reflects established functions of Mediator in transcriptional regulation. Disruption of this axis, through genetic ablation or pharmacological inhibition of CDK8/19, impairs TFE3 fusion dependent transcription and suppresses tumor growth in cellular and in vivo models, identifying the Mediator kinase module as a potential therapeutic target in TFE3 RCC. Statistical significance was determined using an unpaired two-tailed Student’s t-test for two-group comparisons, one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons against a control group, or two-way ANOVA followed by Sidak’s post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: The following primary antibody was used: rabbit monoclonal anti Cyclin C (#68179; CST; 1:200).

Techniques: Quantitative RT-PCR, Derivative Assay, Inhibition, Expressing, Transduction, shRNA, Staining, Flow Cytometry, Fluorescence, Knockdown, Activation Assay, Colony Assay, Control, In Vitro, Knock-Out, Western Blot, Cell Counting, In Vivo, Injection, Immunohistochemical staining, Knock-In, Imaging, Disruption, Two Tailed Test