Fig 2: Brf1 and Pol III genes are mediated by Tam. (A–D): MCF‐7 cells were treated with 12.5 μm Tam (tamoxifen). The cells were treated as described above. The levels of Brf1 protein were measured by western blot (B). RT‐qPCR was performed to determine the amounts of Brf1 mRNA (A) and pre‐tRNAL eu (C) and 5S rRNA transcription (D). The fold changes were calculated by normalizing to the amount of GAPDH mRNA. The bars represent mean ± SE of at least three independent determinations. (E–F): Soft agar assay: MCF‐7 cells were transfected with mm RNA, ERα siRNA, or Brf1 siRNA for 48 h, respectively. The cells were seeded in 6× well plates and treated with ethanol (25 mm) and Tam (12.5 μm) as previously described (Zhong et al., 2014). The cells were analyzed for colony formation in soft agar. Colonies were counted at 2–3 weeks after plating. Values are the means ± SE (n ≥ 3). *P < 0.05 as indicated.

Fig 3: Expression of human BRF1 rescues loss of Brf1 liver phenotype. a IHC for BRF1, p53 and p21 on liver samples from mice harvested at day 8 post-induction. Mouse genotypes are indicated above the panels. WT mice are a combination of AhCre Brf1+/+ mice and AhCre negative mice, Hom mice are AhCre Brf1fl/fl and Hom TG mice are AhCre Brf1fl/fl HprtLSLBRF1 mice containing an extra copy of human BRF1. b Graph depicting mouse liver weight as a fraction of total body weight of animal harvested 8dpi. Genotypes are indicated on the x-axis. WT mice are a combination of AhCre Brf1+/+ mice and AhCre negative mice (n = 11), Hom mice are AhCre Brf1fl/fl (n = 11) and Hom TG mice are AhCre Brf1fl/fl HprtLSLBRF1 mice containing an extra copy of human BRF1 (n = 6). P-values are calculated for a non-parametric Mann–Whitney test. Changes between WT and Hom/TG mice are not significant whereas changes between Brf1fl/fl and WT animals are significant (p < 0.05). c Graphs showing levels of the specified liver enzymes in the blood of mice with the indicated genotypes. WT mice are a combination of AhCre Brf1+/+ mice and AhCre negative mice (n = 8), Hom mice are AhCre Brf1fl/fl (n = 5) and Hom TG mice are AhCre Brf1fl/fl HprtLSLBRF1 mice containing an extra copy of human BRF1 (n = 5). P-values are calculated for a non-parametric Mann–Whitney test. Changes between WT and Hom/TG mice are not significant

Fig 4: High Brf1 expression correlated with a poor prognosis of lung cancer. (a) The IHC staining scores of Brf1 expression. 185 cases in paired different clinical stages of lung cancer tumor tissues show high Brf1 expression, compared to low expression of Brf1 expression corresponding to normal tissues of these cases (N = 185). (b) 226 cases of human lung cancer patients were performed for Kaplan-Meier analysis of the overall survival period. Lung cancer patients (N = 226) with low versus high expression of Brf1 (Kaplan-Meier analysis with the log-rank test, p < 0.01). (c) Histological classification of the 226 cases of lung cancer. (d) ROC curve analysis. The result reveals that patients with high Brf1 expression display short survival times.

Fig 5: Immunohistochemical staining of Brf1 in lung cancer. (a) Comparison of Brf1 staining in tumor foci tissue with paracancer tissue (para-can) of lung cancer patients. Strong staining signals of Brf1 expression are seen in tumor foci of lung cancer (a, left panel). Weak signals of Brf1 staining are detected in para-can tissue of this disease (a, right panel). Top panel: 100x magnification (scale bar = 200 μm); bottom panel: 630x magnification. (b) Comparison of Brf1 IHC and H&E staining in the same cases of lung cancer. IHC staining about the signals of Brf1 expression in both cytoplasm and nuclei of tumor tissues of lung cancer; (b, left panel) H&E staining of tumor tissues of lung cancer; (b, right panel) 100x magnification (scale bar = 200 μm); 400x magnification. (c) Comparison of Brf1 expression in tumor foci with adjacent noncancerous tissue (ANT). The levels of Brf1 expression were detected in four lung cancer lesions (c, upper panel) and their paired ANT (c, lower panel). 400x magnification (scale bar = 50 μm). The results indicate that Brf1 expression was increased in the tumor tissues at different stages of lung cancer, compared to noncancerous tissues, ANT.