Fig. 8

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