Fig. 2.

Upregulation of the transcription factor E2F1 resulting from loss of CBX5 leads to increased EGFRi resistance in vitro and in vivo. (A) Heatmap showing the top 50 genes up-regulated in HCC827 cells expressing CBX5 shRNAs compared to cells expressing an NS shRNA, as measured by RNA-seq. (B) Expression of E2F1 mRNA in the indicated LUAD cell lines expressing a CBX5 shRNAs or NS shRNA, measured by qRT-PCR. Data were normalized to ACTINB, and the results with NS shRNA were set to 1. (C) Immunoblot analysis measuring expression of E2F1 and CBX5 in the indicated LUAD cell lines expressing an NS or CBX5 shRNA. (D) Relative enrichment of CBX5 on the E2F1 and ACTINB promoters, as measured by CUT&RUN. (E) PC9 cells expressing an NS or CBX5 shRNA alone, or in combination with an E2F1 shRNA, were treated with erlotinib (100 nM) or DMSO, and survival was measured in clonogenic assays. Representative wells for cells grown under the indicated conditions are shown. (F) PC9 cells expressing an NS or CBX5 shRNA alone, or in combination with an E2F1 shRNA, were injected subcutaneously into the flanks of NSG mice (n = 5). Mice were treated with either vehicle (0.5% methylcellulose) or erlotinib (25 mg/kg) 3 d per week until the end of the experiment. Average tumor volumes at the end of the experiment are shown. Data are presented as the mean ± SEM. ns = not significant; **P < 0.01, ***P < 0.001, and ****P < 0.0001.