Fig 2: CDYL is downregulated in cervical cancer and associated with poor prognosis in cervical cancer patients. (A) CDYL mRNA expression was decreased in cervical cancer tissues as determined by qRT-PCR (p<0.01). (B) Images of the IHC analyses of CDYL using cervical cancer TMA tissue sections. (C) Violin plot of CDYL expression as determined by the IHC score using TMA tissue sections (p<0.01). (D) Overall survival of the patients with cervical cancer were determined by CDYL mRNA expression level . The difference was compared between low CDYL expression and high CDYL expression group.

Fig 3: AR is colocalized and interacts with CDYL in male testes and Sertoli cells. (A) Immunohistochemical staining of AR and CDYL in testes obtained from wild-type and ARKO mice. Bar = 50 um. (B) Localization of AR and CDYL (upper) and co-localization of AR, CDYL, and DAPI (bottom) in the testes obtained from wild-type mice by immunofluorescence analysis. Bar = 20 μm. (C) Interaction was observed between AR and CDYL in wild-type mouse testes. IgG was used as the control for Western blotting in each group. “Input” means the sample on 10% of volume used for IP. (D) Protein expression patterns of AR and CDYL in wild-type mouse testes and ARKO mice by Western blotting. GAPDH was used as the internal control. (E) AR and CDYL mRNA expressions were detected in testicular tissues between wild-type and ARKO mice by quantitative RT–PCR assay. (n ≥ 3) * p ˂ 0.05, by unpaired two-tailed Student t tests was significant compared with the control. Data are expressed as the mean ± standard error of three samples per group. S: Sertoli cell; L: Leydig cell; M: myoid cell; SP: spermatids.

Fig 4: Characterization of CDYL-regulated Kcr of RPA1.(A) The expression of the indicated proteins was measured by Western blotting in HeLa cells. (B) Immunoprecipitations in WT and CDYL KO HeLa cells with anti-PanKcr or anti–immunoglobulin G (IgG) followed by immunoblotting with antibodies against the indicated proteins. (C) Immunoprecipitations in WT and CDYL KO HeLa cells with anti-RPA1 or anti-IgG followed by immunoblotting with anti-PanKcr or anti-PanKac. (D) Schematic diagram of RPA1. (E) K88, K379, and K595 are major Kcr sites of RPA1 in vivo. Immunoprecipitation assays were performed in HeLa cells overexpressing indicated FLAG-tagged RPA1 constructs with anti-FLAG followed by immunoblotting (IB) with anti-FLAG or anti-PanKcr. (F) The specificity of antibodies against RPA1 K88cr, RPA1 K379cr, and RPA1 K595cr was verified by dot blot assays. The nitrocellulose membrane was spotted with the indicated amounts of uncrotonylated or crotonylated RPA1 peptides and immunoblotted with the indicated antibodies. (G) Verification of the specificity of anti–RPA1-K88cr, anti–RPA1-K379cr, or anti–RPA1-K595cr by Western blotting. Immunoprecipitation assays were performed in HeLa cells overexpressing the indicated FLAG-tagged RPA1 constructs with anti-FLAG followed by immunoblotting with the indicated antibodies, respectively. (H) The Kcr level of RPA1 is increased in CDYL KO cells. WT and CDYL KO HeLa cells were transfected with FLAG-RPA1, followed by immunoblotting with the indicated antibodies. (I) Top: Coimmunoprecipitation assays with lysates from HeLa cells overexpressing FLAG-CDYL using anti-FLAG followed by immunoblotting with antibodies against the indicated proteins. Bottom: Coimmunoprecipitation assays with lysates from HeLa cells using anti-CDYL followed by immunoblotting with antibodies against the indicated proteins. (J) Top: GST pull-down assays with GST-fused CDYL and in vitro transcribed/translated RPA1. Bottom: Coomassie brilliant blue staining of the purified GST and GST-CDYL. (K) HeLa cells were treated with UV (20 J/m2), IR (10 Gy), HU (1 mM), CPT (1 μM), VP16 (40 nM), or dimethyl sulfoxide (DMSO) for 8 hours. Western blotting was performed with the indicated antibodies. Each scale bar represents the mean ± SD for triplicate experiments. Mean data are normalized to RPA1. *P < 0.05 versus lane 1 (two-tailed unpaired Student’s t test). (L) In the presence or absence of CPT treatment, cellular extracts from WT and CDYL KO HeLa cells were immunoblotted with the indicated antibodies.

Fig 5: Effect of the EZH2 inhibitor on CDYL-induced chemoresistance. (A) Western blots showing EZH2 and CDKN1C levels in H69 cells and CDYL-overexpressing H69 cells treated with or without GSK126 (left panel) and quantification of CDKN1C levels (right panel). (B) CCK8 analysis of IC50 values in the H69 cells shown in (A). * P < 0.05, ** P < 0.01, and *** P < 0.001. (C) Effects of chemotherapy with or without GSK126 on tumour growth in mice injected with H69 cells and CDYL-overexpressing H69 cells (n = 5 animals per group). (D) Tumour growth curve for the mice shown in (D). * P < 0.05. (E) Western blots showing CDYL, EZH2 and CDKN1C levels in xenograft tumours.