Fig 2: SIRT7 augments PCAF binding to MDM2.a Stable SIRT7 knockdown (shSIRT7) or control (shCtr) HCT116 cells were transfected with FLAG-PCAF and treated with 10 µM MG132 for 8 h. Cell lysates were subjected to immunoprecipitation with FLAG-conjugated M2 beads before immunoblotting. b HCT116 cells were transfected with the indicated SIRT7 plasmids or an empty vector and treated with 10 µM MG132 for 8 h. Cell lysates were subjected to immunoprecipitation with an anti-MDM2 antibody before immunoblotting. c HCT116 cells were transfected with SIRT7 (si-SIRT7) or negative control (NC) siRNAs and transfected with FLAG-PCAF before being subjected to glucose deprivation (GD) and 10 µM MG132 treated for 12 h. Total cell lysates were immunoprecipitated with FLAG-conjugated M2 beads before immunoblotting. d HCT116 cells were transfected with the indicated FLAG-PCAF plasmids or an empty vector and treated with 10 µM MG132 for 8 h before being subjected to 10 µM MG132 treated with or without glucose deprivation (GD) for 12 h. Total cell lysates were immunoprecipitated with FLAG-conjugated M2 beads before immunoblotting. e HCT116 cells were transfected with the indicated FLAG-PCAF and Myc-Ub plasmids and treated with 10 µM MG132 for 8 h. Total cell lysates were immunoprecipitated with an anti-MDM2 antibody and the ubiquitination levels were analyzed by immunoblotting. f PCAF(WT) and PCAF (KO) HCT116 cells were transfected with the indicated plasmids and then treated with CHX for the indicated time. Cells were harvested and MDM2 protein levels were measured by immunoblotting. g Quantitation of MDM2 protein levels. Immunoblots in f were scanned and normalized to ß-actin. The data represent the means ± SD (n = 3 experiments).

Fig 3: TP increases the binding of RPL23 with MDM2 and activates p53 and p53-regulated proteins. (A) Cell lysates of TP-treated cells (5×106) were immunoprecipitated with anti-MDM2 antibodies, followed by immunoblotting with anti-MDM2, p53 and RPL23 antibodies. For each lysate, 20% of the quantity used for IP was loaded as an input control. (B) Cell lysates from 5×106 A549 cells receiving various treatments for 48 h were subjected to western blot analysis with the indicated antibodies; GAPDH was used as an internal control. (C) Densitometric analysis of protein expression. Data are presented as the mean ± SD of at least three independent experiments. Data were analyzed using one-way ANOVA combined with Tukey's multiple comparisons test. ***P<0.001, ****P<0.0001 vs. control. TP, triptolide; MDM2, mouse double minute 2; RPL23, ribosomal protein L23; PUMA, p53-upregulated modulator of apoptosis, C-Cas, cleaved caspase; p, phosphorylated; IP, immunoprecipitation.

Fig 4: Deficient RYR2 contributes to malignant phenotypes of ESCC. (A) A schematic representation of the domain structure of RYR2 shows the location of somatic variants identified in ESCC tumors. (B) Representative images of IHC for RYR2 in ESCC tissues and matched adjacent normal tissues. Scale bars, 200 µm. (C) Left: Kaplan-Meier survival analysis of patients with ESCC stratified by RYR2 expression (n = 104; p = 0.003, log-rank test). Right: Multivariate analysis of the hazard ratios (HR) showed that the downregulation of RYR2 was an independent prognostic factor for the overall survival (by the multivariate Cox proportional hazard regression model). The HR is presented as the means (95% confidence interval, 95% CI). (D) Left: Kaplan-Meier survival analysis of patients with ESCC stratified by RYR2 localization (n = 104; p = 0.038, log-rank test.) Right: Multivariate analysis of the hazard ratios (HR) showed that the translocation of RYR2 may be an independent prognostic factor for the overall survival rate (by the multivariate Cox proportional hazard regression model). The HR is presented as the means (95% confidence interval, 95% CI). (E) RYR2 expression and localization in normal and tumor tissue. (F) The mRNA levels of RYR2 in YES2 and KYSE150 cells after a knockdown by siRNAs. (G) The protein levels of RYR2, MDM2, and p53 in YES2 (left) and KYSE150 (right) cells after a knockdown by siRNAs. (H) MTS assay on YES2 and KYSE150 cells transfected with siRYR2 or siCtrl. (I) Colony formation assay on YES2and KYSE150 cells transfected with siRYR2 or siCtrl. Up: representative images. Down: statistical analysis. (J) Transwell assays on YES2 and KYSE150 cells transfected with siRYR2 or siCtrl. Left: representative images. Right: statistical analysis.

Fig 5: LLU-206 induces degradation through the MDM2-mediated ubiquitin–proteasomal system. (A) The protein levels of ARfl or ARv7 were detected in 22RV1 cells treated with LLU-206 (5 µmol/L, 10 µmol/L) and ENZ (10 µmol/L) for 48 h. (B) The effect of LLU-206 on ARfl and ARv7 stability was measured by Western blot. The protein levels were detected in 22RV1 cells, which were incubated with CHX (50 µg/mL) with or without LLU-206 for a different time. (C) Statistical analysis of the relative protein expression of ARfl and ARv7 for three independent experiments (n = 3). (D) Western blot assay for the protein levels of ARfl and ARv7 in 22RV1 cells, which were treated with LLU-206 (10 µmol/L, 24 h), and MG132 (10 µmol/L, 6 h). (E) IP was conducted using anti-ARfl and immunoblotting was performed with ARfl, ARv7 and ubiquitin antibodies. (F) IP was conducted using anti-ARv7 and immunoblotting was performed with ubiquitin and ARv7 antibodies. (G) LLU-206 was docked with 63,890 human crystal structure proteins by computer modeling docking studies. The top 9 docking proteins are listed according to their binding energy score. (H) The docking model of LLU-206 (purple), AR-LBD (blue) and MDM2 (green). (I) Binding sites of LLU-206 with MDM2. (J) The ARfl, ARv7 and MDM2 protein levels were tested in 22RV1 cells with the treatment of MDM2 antagonist nutlin3 (5 µmol/L) and LLU-206 (10 µmol/L, 24 h). (K) Co-IP was conducted using anti-ARfl or anti-ARv7. Immunoblotting was performed with MDM2 and ARfl or ARv7 antibodies in 22RV1 cells.