Fig 2: Loss of Rb1 accelerates lung cancer progression in a Kras-driven model of lung cancer. (A) Schematic of experimental design to assess the metabolic function of pRb in Kras-driven lung tumors in vivo. For all experiments, non-instilled KrasLSL/Rb1lox/lox mice served as normal lung controls. (B) Kaplan–Meier survival analysis for Rb1 wild-type (Rb1+/+) (n = 53) or knock-out (Rb1-/-) (n = 47) KrasLSL mice. (C) Representative H&E staining of lung tissue from normal, Rb1+/+, and Rb1-/- mice (n = 3).

Fig 3: Assessing the interaction of Ser5P-PolII, RB1, ELF1, or MAZ with the core CXXC5 promoter elements in cellula. (a) ChIP. Fragmented chromatin from MCF7 cells processed for ChIP was immunoprecipitated with a species-specific IgG or an antibody specific to Ser5P-PolII, RB1, or ELF1. Fragmented chromatin of MCF7 cells transiently transfected with MAZ?N was also processed for ChIP to ensure that the MAZ antibody directed to the carboxyl-terminus of MAZ to be used in ChIP is capable of precipitating the endogenous MAZ. ChIP precipitates were subjected to SDS-PAGE for Ser5P-PolII and RB1 or to SDS-PAGE for ELF1 and MAZ?N followed by IB using antibodies indicated for ChIP. Asterisk (*) indicates the protein of interest. Input, IgG together with heavy (HC) and light (LC) chains of IgG are indicated. Molecular masses (MM) in kDa are denoted. (b) ChIP-qPCR. While identical primer sets for each antibody were used in assessing the interaction of a transcription factor (TF) to Segment A as the core CXXC5 promoter elements (CXXC5) or to the Exon2 of Myoglobin (MB) as a control, distinct primer sets were used for the promoter of a target gene of each TF. The mean ± SE of three independent experiments performed in triplicate is shown. Asterisk (*) indicates significant differences depicted as fold change compared to IgG.

Fig 4: CDK4/6 inhibitor combined with metformin promoted cell cycle arrest.A, B Flow cytometry results showed that the combination of the CDK4/6 inhibitor LY2835219 (HSC3: 0.1 µM; HSC6: 0.3 µM; Cal27: 1.25 µM) with metformin (HSC3: 10 mM; HSC6: 1 mM; Cal27: 10 mM) induced cell cycle arrest in the G0/G1 phase. C Western blot results showed that LY2835219 upregulated the expression of p16 and p21 and downregulated the expression of pRb. D Western blot results showed that metformin upregulated the expression of p16 and p21 and downregulated the expression of pRb. E Compared with LY2835219 monotherapy, the combination treatment significantly upregulated p21 expression and suppressed p-Rb expression. F Immunohistochemistry results (400×) showed that LY2835219 and metformin synergistically upregulated p21 expression and downregulated pRb expression in HSC6 xenograft tumors. Bar: 100 µm. *P < 0.05 when compared with the control group; #P < 0.05 when compared with the combined group; one-way ANOVA. LY: LY2835219; Met: Metformin.

Fig 5: Deletion of RB1 alters UV susceptibility.(A) Western blot of RB1 in RB1 knockout (RB1 KO) and wild-type (WT) IMR-90 cells. Tubulin was used as loading control. Molecular weight (MW) in kD is shown next to blot. (B) Illustration of UV lesion mapping method. Both wild-type and RB1 KO cells were treated with 100 J/m2 UVC. Cells were immediately lysed after UV irradiation and DNA was harvested. Purified DNA was then immunoprecipitation with anti-cyclobutane pyrimidine dimer antibody. Immunoprecipitated fragments were then repaired by photolyases prior to library preparation and Illumina sequencing. (C) Annotation of previously published RB1 peaks in proliferating and quiescent IMR-90 cells (Chicas et al, 2010). (D) Expected versus observed RB1 ChIP-seq peaks in top 10% and bottom 10% UV susceptible genomic regions, in both proliferating and quiescent cells. Expected values were generated based on the null hypothesis that susceptibility change has no association with location of RB1 peaks. Chi-squared test (P < 0.001) indicated that there is significant depletion and enrichment of RB1 peaks in top 10% and bottom 10% regions, respectively.