Fig 2: DAPK3 as a binding partner regulates PBRM1 stability in renal cancer cells.a A schematic diagram depicted that UBE3A contained a consensus DAPK phosphorylation motif which was adjacent to the PKA phosphorylation site. b Western blotting analysis the whole-cell lysates (WCL) of 293T cells. c Western blotting analysis the WCL 786-O and ACHN cells. d Western blotting analysis of UBE3A proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-DAPK3 recombinant proteins. e Western blotting analysis of DAPK3 proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-UBE3A recombinant proteins. f, g 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for Western blotting analysis (f) and RT-qPCR analysis (g). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). ns not significant. h, i 786-O cells were transfected with indicated shRNAs. Seventy-two hours post infection, cells were harvested for western blotting analysis (h) and RT-qPCR analysis (i). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). ns not significant. j 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. k 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. The GAPDH was recognized as the loading control. The protein level of PBRM1 was first normalized to loading control. The normalized values were further normalized to the values in 0 h group. Immunoblots (IB) are representative of results from two independent experiments (n = 2). Statistical significance was determined by multiple student’s t-test at the time point of 5, 10, 15 h. Data presented as Mean ± SEM with two replicates. Ns not significant; ***P < 0.001. Data presented as Mean ± SEM with two replicates. Ns not significant; ***P < 0.001. l 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Fig 3: RBPJ/DAPK3/UBE3A/PBRM1/p21 contributes to the resistance of renal cancer cells to CDK4/6 inhibitors.a analysis of the protein levels of UBE3A in the TMA of patients with renal cell carcinoma. N = 40; ***P < 0.001. b, c 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for RT-qPCR analysis (b) and MTS assay (c). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. d, e 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were harvested for RT-qPCR analysis (d) and MTS assay (e). Statistical significance was determined by Student’s t-test. Data presented as Mean ± SEM with three replicates (n = 3). ***P < 0.001. f The bioinformatics analysis indicated that UBE3A is positively correlate with cell cycle in renal cancer specimens through analyzing the TCGA dataset. g, h 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for the MTS assay (g) and colony formation assay (h) treated with or without palbociclib (20 µM). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. i–k 786-O cells were infected with indicated shRNAs for 72 h. After puromycin selection, cells were harvested and injected subcutaneously into nude mice for xenografts assay and treated with or without palbociclib. The image of tumor was shown in panel i. The tumor mass was demonstrated in panel j. The tumor growth curve was indicated in panel k. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with five replicates (n = 5). **P < 0.01; ***P < 0.001. l 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were collected for the MTS assay after treated with indicated concentration of palbociclib for another 24 h. Statistical significance was determined by Student’s t-test. Data presented as Mean ± SEM with three replicates (n = 3). **P < 0.01; ***P < 0.001. m 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were collected for the MTS assay after treated with indicated concentration of palbociclib for another 24 h. Statistical significance was determined by Student’s t-test. Data presented as Mean ± SEM with three replicates (n = 3). Ns not significant; *P < 0.05; ***P < 0.001. n–p 786-O cells were treated with indicated small inhibitors. Cells were subjected to MTS assay (n) and Xenografts assay (o, p). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For MTS assay, data presented as Mean ± SEM with three replicates (n = 3). For xenografts assay, data presented as Mean ± SEM with five replicates (n = 5). **P < 0.01; ***P < 0.001.

Fig 4: Validation of CRISPR/Cas9-mediated DAPK1 knockout in HCT116 colorectal cancer cells.a Western Blot of DAPK1 expression status in various colorectal cancer cell lines. Representative images of two independent experiments are shown. GAPDH served as loading control. b Immunofluorescence staining of DAPK1 (green) in parental HCT116 cells and DAPK1 ko clones. Cells were counterstained with phalloidin for F-actin (red) and nuclear Hoechst (blue). Fluorescence microscopy was performed using a x 100 oil immersion objective. Representative images of two independent experiments are shown. Scale bar = 20 µm. c Protein expression of DAPK1 family members DAPK1, DAPK2, DAPK3, DRAK1, DRAK2 and DAPK1 phosphorylation target pMLC in HCT116 wildtype cells and DAPK1 ko clones were detected by Western Blotting using specific primary antibodies. Representative images of two independent experiments are shown. * images were cropped here; all samples were analyzed on the same SDS-PAGE gel; ** GAPDH blot has been used twice see Fig. 5b, proteins have been loaded on the same membrane. d Western Blot analysis of stem cell markers (CD133, CD44) and EMT markers (epithelial marker: E-cad = E-cadherin, mesenchymal marker: Vimentin). Representative images of two independent experiments are shown.*images were cropped here; all samples were analyzed on the same SDS-PAGE gel. e Representative images of endogenous phospho-ERK1/2 (red) levels of immunostained HCT116 cells and DAPK1 ko clones examined by confocal immunofluorescence microscopy (63x; enlarged: cropped and zoomed in). Cells were nuclear counterstained with Hoechst (blue). Immunofluorescence was repeated in two independent experiments and representative images are shown. White arrows: empty nucleus; dashed arrow: nuclear expression of pERK1/2. Scale bar = 50 µm. f pERK1/2 expression analyzed by Western Blot in cytoplasmic (C) and nuclear (N) protein fractions. Representative images of two independent experiments are shown. GAPDH served as loading control in total and cytoplasmatic protein fractions. Lamin A/C was used for nuclear loading control.

Fig 5: A hypothesis model depicted that PKA phosphorylated UBE3A to prevent UBE3A degrading PBRM1.DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.