Fig. 4: RB1-deficiency activates the E2F-dependent transcription of stathmin.

a Transcriptome profiling of A549 RB1^+/+ and RB1^−/− cells and the gene ontology (GO) analysis by molecular functions for differentially expressed genes between the isogenic cells. Fisher Exact test was performed to categorize the genes in GO groups (P < 0.05). Among the differentially expressed genes, tubulin binding proteins (total 10) are shown in the circles as up-regulated (red) or down-regulated (blue) genes in RB1^−/− cells. b Validation of the expression of tubulin binding proteins with RT-qPCR analysis (mean ± SEM, n = 3 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001 between two indicated groups, determined using one sample t test. n.s denotes not significant. c ChIP-Atlas database analysis to predict the E2F1 target genes among the tubulin binding proteins. d, e Up-regulation of stathmin (STMN1) level and reciprocal down-regulation of α-tubulin level in RB1^−/− lung cancer cells. A549 (d) or HCC827 (e) RB1^+/+ and RB1^−/− cells were analyzed for stathmin and α-tubulin protein levels. f A549 RB1^+/+ cells were transfected with RB1 siRNA and analyzed for stathmin and α-tubulin protein levels. g A549 RB1^−/− cells were transfected with E2F1 siRNA and analyzed for stathmin protein level. ChIP analysis of the STMN1 promoter in A549_RB^−/− cells using a normal mouse IgG, anti-E2F1 (α-E2F1) (h), anti-E2F2 (i) or anti-E2F3 (j) antibody and the primer sets specific for a predicted E2F1, E2F2 or E2F3 binding site on the promoter of human stathmin (STMN1 promoter) or the 10 kb 5’-upstream of the stathmin transcription start site (Neutral region) as a background. Data are presented as percent chromatin precipitated by IgG and E2F1 antibodies at both locations (mean ± SEM, n = 3 independent experiments). *P < 0.05; **P < 0.01 between two indicated groups, determined using two-sided Student’s t test. k The protein levels of RB1, stathmin and α-tubulin in a panel of lung cancer cell lines. l, m The RB1 and stathmin protein expression levels from 365 lung cancer patients were downloaded from TCGA project archived in LinkedOmics (http://www.linkedomics.org/). The data were shown in the heat map (l) and volcano plot (m), and stathmin is indicated with the red arrows.