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. Author manuscript; available in PMC: 2012 Mar 15.
Published in final edited form as: Oncogene. 2011 Apr 4;30(37):3943–3952. doi: 10.1038/onc.2011.105

Fig. 6. Reduction of Brf1 and H3S28ph represses EGF-induced cell transformation.

Fig. 6

(A) Repression of Brf1 expression inhibits EGF-induced Pol III gene transcription. JB6 cells were transfected with mismatch (mm) RNA or Brf1 siRNAs for 48 h and treated with EGF. Cell lysates and RNA were isolated from these cells. Immunoblot analysis was performed to determine cellular level of Brf1 protein (left) and pre-tRNALeu (middle), 5S rRNA (right) and GAPDH transcripts were measured by RT-qPCR. (B) Down-regulating Brf1 expression decreases EGF-induced anchorage-independent growth. JB6 cells expressing Brf1 siRNAs were poured in triplicate into 6-well plate with 0.35% agar containing 0ng or 20ng/ml EGF. The cells in A were analyzed for growth in soft agar. (C) Blocking H3S28ph signaling represses EGF-induced cell transformation. JB6 stable cell lines expressing pcDNA3 vector (vector), pcDNA3-wild type H3 (WT H3) or pcDNA3-H3S28A (H3S28A) were poured into 6-well plate with EGF as designated at (B). Cells in (A) and (B) were be incubated at 37°C in 5% CO2 for 1-2 weeks and were fed with fresh complete media with or without EGF twice weekly. Colonies were counted at 1-2 weeks after plating. Values are the means ± SEM (n ≥ 3).