Fig 2: High expression levels of CDK6 confers augmented IMiD-resistance.A Overexpression of CDK6 in MM.1S cells using lentiviral transduction confirmed through western blot analysis. B Cell viability of CDK6-overexpressing MM.1S cells upon 96 h treatment with lenalidomide and pomalidomide at indicated concentrations. (N = 3 biologically independent replicates). C Overexpression of CDK6 in OPM2 cells using retroviral transduction confirmed through western blot analysis. D Cell viability of CDK6 WT or K43M-overexpressing OPM2 cells upon 96 h treatment with lenalidomide and pomalidomide at indicated concentrations. (N = 3 biologically independent replicates) Control denotes empty vector. Cell viability is normalized to respective DMSO conditions. Data represent the mean ± SD of biological triplicates. Source data are provided as a Source Data file.

Fig 3: miR-486 can inhibit the proliferation of osteosarcoma cells. (A) After overexpression of miR-486, MG63 and U2OS cell proliferation was detected by MTT assay. Data are shown as mean ± SEM. (B) After inhibiting the expression of miR-486, MG63 and U2OS cell proliferation was detected by MTT assay. Data are shown as mean ± SEM. (C) after transfecting miR-486 mimic/control in MG63 cells, the protein level of CDK4 and CDK6 were detected by western blot analysis. Data are shown as mean ± SEM. **P<0.05. (D) After transfecting miR-486 mimic/control in MG63 cells, the mRNA level of CDK4 and CDK6 were detected by real-time PCR. Data are shown as mean ± SEM. **P<0.05. (E) After transfecting miR-486 inhibitor/control in MG63 cells, the protein level of CDK4 and CDK6 were detected by western blot analysis. Data are shown as mean ± SEM. **P<0.05. (F) After transfecting miR-486 inhibitor/control in MG63 cells, the mRNA level of CDK4 and CDK6 were detected by real-time PCR. Data are shown as mean ± SEM. **P<0.05.

Fig 4: CEP164 depletion accelerates the cell cycle by elevation of Cyclin D-CDK6. (A) The indicated Panc1 cells were cultured in serum-fed medium for 48 h and analyzed using FACS. The proportion of cells at each cell cycle stage was determined. The average of three independent experiments is shown. (B,C) The indicated Panc1 cells were cultured in serum-fed medium for 48 h. (B) Relative amounts of the indicated mRNA were determined using quantitative PCR. GAPDH was used as a control. The average of four to eight independent experiments is shown. (C) Cell extracts were immunoblotted with indicated antibodies. β-Actin was used as a loading control. The amount of indicated proteins was quantified using ImageJ, and the relative values and GLI2FL/GLI2R ratio are shown below the image. (A,B) All data are shown as mean ± SEM. *p < 0.05 compared with Cep164-1 + EV (A) (Chi-squared test), compared with WT + EV and Cep164-1 + Cep164 (B) (two-tailed Student’s t-test). Uncropped images of western blots are shown in Supplementary Figure 5.

Fig 5: CDK6 EE LT-HSCs Outcompete Wild-Type HSCs without Exhaustion(A–D) NSG mice were injected with sorted Lin− CD34+ CD38− cells transduced with CDK6 EE or control (LUC) lentiviral vectors (GFP+ cells) and untransduced competitive cells (GFP−). Bone marrow was harvested at indicated time points post-transplantation and analyzed by flow cytometry. (A) Percentage of GFP+ cells among engrafted human hematopoietic cells (CD45+). Time 0 corresponds to percentage of GFP+ cells before injection in four independent CB samples. 4 weeks post-transplantation: n = 13 LUC and 14 CDK6 EE mice; 20 weeks post-transplantation: n = 25 LUC and 23 CDK6 EE mice. (B) Lymphoid to myeloid ratio (percentage of CD19+/CD33+) among GFP+ cells at 20 weeks post-transplantation. n = 25 LUC and 23 CDK6 EE mice. In (A) and (B), boxplots represent median, 25th, and 75th percentiles and whiskers represent min and max. Gray boxes, LUC; white boxes, CDK6 EE. (C) Percentage of GFP+ cells among LT-HSCs at 20 weeks post-transplantation. (D) Absolute number of LT-HSCs at 20 weeks post-transplantation. In (C) and (D), n = 6 mice from two CB samples. Individual mice, median, and interquantile range are shown. In (A)–(D), ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01 by Mann-Whitney test.(E and F) CDK6 EE LT-HSCs expand over serial transplantation. LUC, CDK6 EE (GFP+), or untransduced (GFP−) LT-HSCs were sorted from primary transplanted mice (n = 2 pools of three to five mice) and injected at four different doses into secondary NSG mice. (E) Engraftment levels (percentage of CD45+ cells) 12 weeks after secondary transplantation (>0.01% CD45+ GFP+ or CD45+ GFP−) at the two highest doses (200 and 400 cells/mouse). Individual mice, median, and interquantile range are shown. (F) Summary table of number of mice engrafted at each dose tested and estimation of LT-HSC frequencies in each group by the ELDA statistical method. See also Figure S5.