Fig 2: A dual BRD4/PLK1 inhibitor sensitizes PC cells to the anticancer effects of the CDK4/6 inhibitor partially through CBX3. A) C4‐2 and PC‐3 cells were transfected with indicated shRNAs for 72 h. Cells were treated with a serial dose of Palbociclib for 24 h and subjected to CCK8 assay. B) C4‐2 and PC‐3 cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with or without 1 µM volasertib for 24 h. Cells were treated with a serial dose of Palbociclib for 24 h and subjected to CCK8 assay. C) C4‐2 cells were transfected with indicated shRNAs for 72 h. Cells were treated with a serial dose of Palbociclib and subjected to colony formation assay. D and E) C4‐2, PC‐3, and DU145 cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with a serial dose (0, 2, 10 µM) of BI‐2536 for 24 h and subjected to Western blot analysis and RT‐qPCR assay. Data presents as mean ± SEM with three replicates. Ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. F and G) C4‐2 cells were treated with or without 10 µM BI‐2536 for 24 h. Cells were subjected to RNA‐seq analysis. Analysis of co‐regulated genes of CBX3 and BI‐2536 treatment. H and I) C4‐2 cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with or without 10 µM BI‐2536 for 24 h. Cells were harvested for Western blot analysis and CCK‐8 assay. J—L) C4‐2 cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were injected subcutaneously into the nude mice for xenograft assay. After the tumor volume approximately reached to 100 mm3, Palbociclib HCl (150 mg Kg−1) or Vehicle (Water) were administrated orally once every other day, Vehicle or BI‐2535 (50 mg Kg−1) were intraperitoneal injected once every other three day. The tumor image was shown in panel J, the tumor mass was shown in panel K, the growth curve was shown in panel L. Data presents as mean ± SEM with six replicates. *, P < 0.05; ***, P < 0.001. M and N) C4‐2 Palbociclib‐resistance cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were injected subcutaneously into the nude mice for xenograft assay. After the tumor volume ≈reached to 100 mm3, Palbociclib HCl (150 mg Kg−1) or Vehicle (Water) were administrated orally once every other day, Vehicle or BI‐2535 (50 mg Kg−1) were intraperitoneal injected once every other three day. The tumor image was shown in panel (M), the growth curve was shown in panel (N). Data presents as mean ± SEM with six replicates. Ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3: PLK1 phosphorylates CBX3 to promote CBX3 binding to RB1 in PC. A) analysis of mass spectrometry of CBX3. B and C) C4‐2 and PC‐3 cells were harvested for IP assay. D) analysis of the Scansite web tool (https://scansite4.mit.edu/#home) and PhosphoSitePlus web tool (https://www.phosphosite.org/). E) the CBX3 WT and CBX3 T60A proteins were translated in vitro. These proteins were purified by Protein G agarose and primary antibodies (Flag‐tag antibody). Then, the proteins were incubated with PLK1 and ATP‐γ‐S and subjected to Western blot analysis. F and G) C4‐2 cells were transfected with indicates constructs or reagents for 48 h. Cells were harvested for IP assay and Western blot analysis. H—J) C4‐2 cells were transfected with indicates plasmids for 48 h. Cells were subjected to RNA‐seq analysis. K–M) C4‐2 and PC‐3 cells were transfected with indicates plasmids or siRNAs for 48 h. Cells were subjected to Western blot analysis and RT‐qPCR analysis. Data presents as mean ± SEM with three replicates. ns, not significant; ***, P < 0.001. N and O) C4‐2 and PC‐3 cells were treated with or without 1 µM volasertib for 24 h. Cells were subjected to Western blot analysis and RT‐qPCR assay. Data presents as mean ± SEM with three replicates. **, P < 0.01; ***, P < 0.001. P) analysis of TCGA dataset to showed the correlation between PLK1 and MCM. P values as indicated. Q—S) C4‐2 cells were transfected with indicated constructs or treated with indicated reagent for 48 h. Cells were harvested for IP assay. T) a model depicting that BRD4 transcriptionally increases the expression of CBX3, the up‐regulated CBX3 was phosphorylated by the PLK1 and bound with RB1 to release E2F1.

Fig 4: Clinical relevance between TRIM26, GPX4 and PLK1.a IHC staining of 83 human glioma samples. Representative images of two specimens are shown. Scale bars, 100 µm. b Correlation of TRIM26, GPX4 and PLK1 expression in (a); Chi-square test. c–e Kaplan-Meier curves showing the overall survival of 83 glioma patients divided based on TRIM26 (c), GPX4 (d) and PLK1 (e) expression; log-rank test. f The mechanistic scheme of this study.

Fig 5: PLK1-mediated TRIM26 phosphorylation increases GPX4 stability.a, b T98G (a) and U118 (b) cells transfected with control siRNA or PLK1 siRNA were subjected to IP and IB with the indicated antibodies. c, d T98G (c) and U118 (d) cells treated as indicated were subjected to IP and IB with the indicated antibodies. e PLA assay between TRIM26 and GPX4 in U118 cells treated with onvansertib (2 nM) or MLN0905 (2 nM). Scale bar: 20 μm. f, g Flag-TRIM26 WT, Flag-TRIM26 S127A or Flag-TRIM26 S127D was co-transfected with Myc-GPX4 into HEK293T cells. After treated with MG132 (20 µM) for 6 h. Cell lysates were subjected to d-IP and IB with the indicated antibodies. h Purified Flag-TRIM26 WT, Flag-TRIM26 S127A or Flag-TRIM26 S127D was incubated with GST or GST-GPX4 conjugated to beads. Pull-down samples and 5% of the input were analyzed by IB. Purified GST and GST-GPX4 were determined by CBB. i HEK293T cells transfected as indicated were subjected to d-IP with anti-Myc antibody and then analyzed by IB. j HEK293T cells treated as indicated were subjected to d-IP with anti-Myc antibody and then analyzed by IB. k HEK293T cells transfected as indicated were subjected to d-IP with anti-Myc antibody and then analyzed by IB. l Flag-TRIM26 WT, Flag-TRIM26 S127A or Flag-TRIM26 S127D was co-transfected with Myc-GPX4 into HEK293T cells. After treated with CHX for the indicated times, then cell lysates were subjected to IB. Quantification of GPX4 levels relative to β-actin is shown; student’s t test; **P < 0.01.