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. Author manuscript; available in PMC: 2016 Mar 26.

Published in final edited form as: Oncogene. 2014 Apr 21;34(13):1698–1708. doi: 10.1038/onc.2014.102

MUC4 knockdown induces histone modification to modulate cyclin E expression. (a) Western blot analysis of lysates from control and MUC4 KD SCC1 and SCC10B cells using specific antibodies recognizing acetylated and methylated histones. (b) Enhanced interaction between pRb and HDAC1/2 in MUC4 KD cells. Association of pRb and HDAC1/2 was studied in the lysates of control and MUC4 KD SCC1 and SCC10B cells by immunoprecipitation and western blotting as described in Materials and Methods. HDAC1 and 2 were immunoprecipitated using specific antibodies and the bound levels of pRb co-precipitated with HDAC1/2 were determined by western blot analysis. (c) Trichostatin A (TSA) treatment induces cyclin E expression and increases SA-β-gal stained cells. MUC4 KD SCC1 and SCC10B cells were treated with TSA (5μΜ) and 48 h and protein lysates were analyzed by western blot analysis and probed with cyclin E, cyclin D1 and acetylated H3K9 specific antibodies. β-actin was used as a loading control. Similarly, TSA treated cells were analyzed for SA-β-gal staining and senescent cells were observed and counted. (d) Decreased H3K9 acetylation at cyclin E promoter. Cross-linked, sheared chromatin was prepared from control and MUC4 KD SCC1 and SCC10B cells and subjected to immunoprecipitation with the H3K9 acetylated antibody. The immunoprecipitated complexes were subjected to PCR analysis using primer pairs spanning the human cyclin E promoter. Isotypic igG Ab was used as negative control, whereas chromatin obtained before immunoprecipitation was used as internal control.

MUC4 knockdown induces histone modification to modulate cyclin E expression. (a) Western blot analysis of lysates from control and MUC4 KD SCC1 and SCC10B cells using specific antibodies recognizing acetylated and methylated histones. (b) Enhanced interaction between pRb and HDAC1/2 in MUC4 KD cells. Association of pRb and HDAC1/2 was studied in the lysates of control and MUC4 KD SCC1 and SCC10B cells by immunoprecipitation and western blotting as described in Materials and Methods. HDAC1 and 2 were immunoprecipitated using specific antibodies and the bound levels of pRb co-precipitated with HDAC1/2 were determined by western blot analysis. (c) Trichostatin A (TSA) treatment induces cyclin E expression and increases SA-β-gal stained cells. MUC4 KD SCC1 and SCC10B cells were treated with TSA (5μΜ) and 48 h and protein lysates were analyzed by western blot analysis and probed with cyclin E, cyclin D1 and acetylated H3K9 specific antibodies. β-actin was used as a loading control. Similarly, TSA treated cells were analyzed for SA-β-gal staining and senescent cells were observed and counted. (d) Decreased H3K9 acetylation at cyclin E promoter. Cross-linked, sheared chromatin was prepared from control and MUC4 KD SCC1 and SCC10B cells and subjected to immunoprecipitation with the H3K9 acetylated antibody. The immunoprecipitated complexes were subjected to PCR analysis using primer pairs spanning the human cyclin E promoter. Isotypic igG Ab was used as negative control, whereas chromatin obtained before immunoprecipitation was used as internal control.