MOESM2 of NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2

Additional file 2: Figure S1. NOS1 blocks IFNα-stimulated gene induction. a SKOV3 and SW480 cells were treated with IFNα for 6 h in the presence or absence of simultaneous GSNO. The mRNA expression of ISGs were analyzed by RT-PCR. b Control/NOS1 (SKOV3, SW480) cells were incubated with IFNα for 6 h, followed RT-PCR analysis. Figure S2. S-nitrosyltion of HDAC2 does not affect its expression. a Control/NOS1 (SKOV3) cells were treated with IFNα (1000 U/ml) for 6 h and 12 h, the expression of HDAC2 was detected by RT-PCR and western blotting. b Control/NOS1 (SKOV3, B16) cells were stimulated with or without IFNα for 6 h. Protein extracts were subjected to the biotin-switch assay. Figure S3. HDAC2 regulates the acetylation status of H4K16. a Densitometric analysis of the data in Fig. 4g (n=3). b A375 cells were transfected si-RNA for 24 h and treatment with IFNα for 1 h. ChIP assays were performed after chromatin was immunoprecipitated with an anti-H3ac antibody. IP chromatin was subjected to qPCR. ns, not significant.

附加文件2：图S1。一氧化氮合酶1（NOS1）抑制干扰素α（IFNα）刺激的基因诱导。a 在同时存在或不存在GSNO的条件下，用IFNα处理SKOV3与SW480细胞6小时，通过逆转录聚合酶链式反应（RT-PCR）分析干扰素刺激基因（ISGs）的mRNA表达水平。b 转染空载对照/过表达NOS1的SKOV3、SW480细胞用IFNα处理6小时，随后进行RT-PCR分析。
图S2。组蛋白去乙酰化酶2（HDAC2）的S-亚硝基化修饰不影响其蛋白表达。a 用1000 U/ml的IFNα处理转染空载对照/过表达NOS1的SKOV3细胞6小时和12小时，通过RT-PCR与免疫印迹实验（western blotting）检测HDAC2的表达水平。b 分别用IFNα处理或不处理转染空载对照/过表达NOS1的SKOV3、B16细胞6小时，收集蛋白提取物进行生物素切换实验（biotin-switch assay）。
图S3。HDAC2调控组蛋白H4第16位赖氨酸残基（H4K16）的乙酰化状态。a 对图4g中的实验数据进行灰度密度分析（n=3）。b 将A375细胞转染小干扰RNA（si-RNA）24小时后，用IFNα处理1小时，使用抗乙酰化组蛋白H3抗体进行染色质免疫沉淀实验（ChIP assays），随后对免疫沉淀的染色质进行实时定量聚合酶链式反应（qPCR）。ns，无统计学显著性差异。