Fig. 9

Knockdown of Beclin-1 (BECN1) in Tet-On-PADI2 Jurkat T cells inhibits autophagy, prolongs Th17-like T cell activation, and increases cell survival. The Tet-On-PADI2 Jurkat T cells with shBECN1 plasmids were treated with 50 µM Dox; “shLuc” was a scrambled RNA knockdown control. A Proteins extracted from Tet-On-PADI2 and Tet-On-Vector cells were detected using antibodies against Beclin-1, cleaved-caspase 9, Apaf-1, cytosolic cytochrome c (Cyt-c), mitochondrial Cyt-c and β-actin. The cells were treated with 50 µM Dox for 48 h. B The bar graph illustrates the percentages of shBECN1-Tet-On-PADI2 cells with autophagosomes. The data are presented as the mean ± SEM of three separate experiments (***p < 0.001). The p value in was calculated by one-way ANOVA. C Proteins extracted from shBECN1-Tet-On-PADI2 cells were detected using antibodies against PADI2, BECN1, LC3B, Atg5–Atg12, and β-actin. D Proteins extracted from shBECN1-Tet-On-PADI2 cells were detected using antibodies against Bcl-xL, cleaved-caspase 3, cleaved-PARP, and β-actin. E RT-PCR was used to determine the cytokine levels of IL-17A, IL-17F, IL-22, IL-21, IL-6, IL-13, IFNγ, and IL-10, and β-actin mRNA expression at 0.5 h, 12 h, 24 h, and 48 h. The value indicates the ratio of protein signal to β-actin signal