Fig 2: Depletion of SIRT1 or BMI1 during replication stress increases TRCs. (A) Depletion of SIRT1 or BMI1 increases replication stress. (Top) (A) Schematic showing how replication speed was measured using DNA fiber analysis. U2OS cells were pre-treated with 0.2 mM hydroxyurea for 4 h then sequentially pulse-labeled with CldU (green) and IdU (red) for 20 min each in the presence of hydroxyurea. Representative DNA fibers show an elongating fork and a stalled fork (bottom, left). The length of the IdU (red) fibers in the elongating fork was measured using ImageJ software and used to calculate the speed of the replication fork. The scatterplot shows the quantification of the speed of replication fork from at least 200 fibers (bottom right). (B) Recruitment of SIRT1 (top) and BMI1 (bottom) to stalled forks was analyzed by PLA assays. U2OS cells were treated with or without (no) 2 mM hydroxyurea for 4 h; then, SIRT1/EdU PLA assays and BMI1/EdU PLA assays were performed as previously described. Left, representative images. Right, quantification of the average number of PLA foci per nucleus. (C) Parental (WT), SIRT1-KO, and BMI1-KO U2OS cells expressing vector control (ctrl) or the indicated shRNA were treated with 0.5 mM hydroxyurea for 12 h, then PLA between PCNA and RNA pol II was performed. Left, representative images of PLA foci (green). Right, the scatterplot shows the quantification of the PLA signal per nucleus from at least 100 cells. (D and E) The cells used and treated as described in panel (C) were subjected to immunostaining. Left, representative immunostaining images. Right, quantification of S9.6 nuclear signal intensity following nucleolar signal removal. In all panels, data are plotted from three independent experiments with means ± SD. The P-values are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001., n.s., not significant; Mann–Whitney test; scale bar, 10 µm.

Fig 3: BMI1 regulates MM-MFs proliferation.A Left schematic shows the cross-breedings performed to generate BMI1fl/flMx1-Cre mice and inducible Cre-mediated disruption of BMI1 in BMI1fl/flMx1-Cre mice after poly I:C administration. BMI1fl/fl mice received poly I:C were designated as wild-type (wt) controls. Western blot showed the disruption of BMI1 protein in MFs from BMI1-KO mice after poly I:C administration (right panel). B Flow cytometry analyzed the expression of CD206 on the surface of cultured MFs and MM-MFs from wt control or BMI1-KO mice. MFI of CD206 indicated for each sample. C CFSE cell proliferation assay indicated the knockout of BMI1 in MM-MFs impaired their proliferation capacity. D Flow cytometry analyzed BMI1 and Ki67 expression of MFs from BM aspirates of 28 MM patients. Left, histograms of BMI1 and Ki67 expression of 2 representative patients with relative lower or higher BMI1 expression. Right panel, the co-expression pattern of BMI1 and Ki67 was showed in dot plots. The correlation was analyzed by linear regression with R square calculated. E The RNA-seq data of murine BM-derived MM-MFs from wt and BMI1-KO mice were analyzed with GSEA, showing enrichment of Hallmark E2F targets and G2M checkpoint pathways in wt MM-MFs. F Heatmap illustrated the RNA-seq data of CDKN2A, showing significant upregulation of CDKN2A in BMI1-KO MM-MFs. G RT-qPCR showed the mRNA expression of P16 was significantly higher in BMI1-KO MM-MFs, relative to wt MM-MFs (left column). Flow cytometry showed the protein level of P16 was increased in BMI1-KO MM-MFs (right panel). **p < 0.01 (Student’s t-test, comparing 2 samples, error bars are SD).

Fig 4: Hedgehog-Myc axis regulates BMI1 overexpression in MM-MFs.A Illustration of direct and trans-well co-culture of MFs and MM cells. B Western blot showed in both direct and trans-well co-culture methods, the expression of BMI1 was elevated in MM-MFs, relative to MFs. C Western blot showed that protein levels of BMI1 were increased in MFs upon rmSHH treatment. D Western blot showed that the GLI1/GLI2 transcriptional activity inhibitor GANT61 suppressed expression of BMI1 in both murine BM-derived and human PBMC-derived MM-MFs. E Western blot showed that the Smo antagonist Cyclopamine reduced expression of BMI1 in both murine BM-derived and human PBMC-derived MM-MFs. F Western blot showed c-Myc expression was increased in both murine and human derived MM-MFs. G Western blot showed that the c-Myc inhibitor 10058-F4 reduced expression of BMI1 in both murine and human MM-MFs. H ChIP-qPCR showed the relative occupancy of c-Myc and IgG control to BMI1 promoter region in MFs and MM-MFs derived from mouse and human. I Schematic of Hedgehog-c-Myc axis regulates BMI1 expression in MM-MFs.

Fig 5: BMI1 regulates pro-angiogenic function of MM-MFs.A Western blot showed the increased expression of MMP2 and VEGF in MM-MFs derived from both mice and human, relative to MFs. B Western blot showed that knockout of BMI1 impaired the expression of MMP2 and VEGF in MM-MFs. C ELISA assay detected the concentration of VEGF in culture supernatant of murine BM-derived M?s and MM-M?s from wt or BMI1-KO mice. Statistical significance was determined by two-tailed Student t-test between wt and BMI1-KO MM-M?s, **P < 0.01. D BMI1 knockout lowered the levels of NO in culture supernatant of MM-M?s. Statistical significance was determined by two-tailed Student t-test between wt and BMI1-KO MM-M?s, *P < 0.05. E GSEA of RNA-seq data showed the enrichment of genes negatively regulating blood vessel endothelial cell migration in BMI1-KO MM-M?s.