Fig 2: Knockdown of HDAC9 increases acetylation and inhibits ubiquitin–proteasomal degradation of RUNX3. A, B The mRNA and protein level of RUNX3 in Lt.shHDAC9-infected NP cells. C Double fluorescence staining of HDAC9 and RUNX3 in isolated NP cells. Scale bar, 50 μm. D NP cells were immunoprecipitated with anti-RUNX3 to analyze of the interaction of HDAC9 and RUNX3. E The acetylation of RUNX3 in HDAC9 knockdown cells. F Lt.shHDAC9-infected cells were treated with MG132 for 8 h and immunoprecipitated with anti-RUNX3 to detect ubiquitination of RUNX3 using anti-ubiquitin (ubi) antibody. G Lt.shHDAC9-infected cells were treated with cycloheximide (CHX) for indicated time points, and then RUNX3 remaining protein level was detected by western blot. H NP cells were infected with Lt.shHDAC9 and Lt.shRUNX3 for 72 h and the protein level of RUNX3 was detected. I Cell viability of NP cells was measured by CCK-8 assay. J Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. K The protein level of p53, p21, PUMA and Cyclin D1 were detected by western blot. Data are represented as mean ± SD (n = 3). A p value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Fig 3: High glucose, fatty acid, and pro-inflammatory cytokine induce oxidative stress and mitochondrial dysfunction in HK-2 cells.A Representative images of reactive oxygen species (ROS)-induced fluorescence in DCFH-DA stained HK-2 cells cultured under different treatments as indicated (magnification, ×100). B Mitochondrial membrane potential of HK-2 cells under various treatments assessed by flow cytometry after JC-1 staining. C Quantitative ROS-induced fluorescence in HK-2 cells; **P < 0.01 versus LG; ##P < 0.01 versus HG; &&P < 0.01 versus HG + PA; n = 4 each group. D Quantitation of mitochondrial membrane potential based on flow-cytometry analysis of JC-1-stained cells. *P < 0.05 versus LG; ##P < 0.01 versus HG; &&P < 0.01 versus HG + PA; n = 4 each group. E Representative confocal microscopic images of HK-2 cells stained with MitoTracker Red. F, G Representative western blots (F) and densitometric quantitation (G) of ATP-citrate lyase (ACL), HMGCR, DGAT1, and TNF-α proteins in HK-2 cells with various treatments. H, I Representative western blots (H) and densitometric quantitation (I) of total cellular p-Drp1, Drp1, and Gp91 proteins. J, K Representative western blots (J) and densitometric quantitation (K) of cytosolic (C) Cyto-c, PUMA, and caspase 3. *P < 0.05, **P < 0.01 versus LG; #P < 0.05, ##P < 0.01 versus HG; &P < 0.05, &&P < 0.01 versus HG + PA; n = 4 each group.

Fig 4: BBC3 expression levels in endometrial tissues and ESCs. Reverse transcription-quantitative PCR was performed to determine BBC3 mRNA expression levels in (A) endometrial tissues and (B) ESCs. Western blotting was performed to determine BBC3 protein expression levels in (C) endometrial tissues and (D) ESCs. **P<0.01 vs. normal tissue; ##P<0.01 vs. normal ESCs. BBC3, Bcl-2 binding component 3; ESC, endometrial stromal cells.

Fig 5: Induction of PUMA contributes to shikonin induced necroptosis. (A) The expression of indicated Bcl-2 family proteins in T24 parental and resistant cells treated with 25 µM cisplatin (Cis). (B) The expression of indicated proteins in T24 resistant cells treated with 0.4 μM shikonin (SKN) and 25 µM cisplatin (Cis) alone or their combination (C+S) for 24 h. (C) T24 resistant cells were transfected with PUMA siRNA, and subsequently treated with 0.4 μM shikonin and 25 µM cisplatin alone or their combination for 24 h. The expression of PUMA and p-MLKL was analyzed by western blot. (D, E, and F) The T24 resistant cells were treated as in (C). D, the cell viability was analyzed by MTT assay. E, the apoptosis was analyzed by Hoechst 33258 staining. F. Crystal violet staining for the cell viability. N, p>0.05; *, p<0.05; **, p<0.01.