Fig. 6

Regulation of Lkb1 influences Treg function by altering the mevalonate pathway in Tregs. A, B Determination of gene expression levels in Tregs treated by shRNA or transduced with lentivirus carrying Lkb1 complementary DNA at 48 h by qPCR analysis and western blots with indicated antibodies. n = 3. C, D Cytotoxic activity of activated CD8⁺CTLs cocultured with Tregs from healty donors (N-Treg) or Tregs with Lkb1 knock down (Sh-Treg) or overexpression (OE-Treg) toward CellTraceTM Far Red-labelled SU-DHL4 cells at the indicated E/T ratios of 10:1. The percentage of PI⁺ cells was used to calculate the % lysis of target cells by CD8⁺ CTLs. n = 3. E Proliferation of CD8⁺ CTLs was determined by coculturing Tag-it Violet labeled CD8⁺ CTLs with Tregs for 3 days. F mRNA expression of mevalonate pathway genes of Tregs with Lkb1 knock down or overexpression. n = 3. G PI staining of SU-DHL4 cells from the co-culture system of CD8⁺CTLs with Tregs from healty donors or with HMGCR or HMGCS interfered. n = 3. H PI staining of SU-DHL4 cells from the co-culture system of CD8⁺CTLs with Lkb1 overexpressed and HMGCR or HMGCS interfered Lkb1 overexpressed Treg cells. n = 3. I The quantity of HMGCR enzyme of Tregs were evaluated by Elisa with Lkb1 knock down or overexpression, as well as HMGCR interfered Lkb1 overexpressed Tregs. n = 3. *P < 0.05; **P < 0.01, ***P < 0.001. Asterisk indicates significant difference from control group as assessed. Pound sign indicates significant difference from different group. In A, p values were determined by two-sided unpaired t-test; In D and G-I, p values were determined by one-way ANOVA; In F, p values were determined by two-way ANOVA; data are presented as mean values ± SEM