Extended Data Fig. 9: LKB1 antagonizes OPA1 deficiency independent of mTOR and AMPK.
From: An LKB1–mitochondria axis controls TH17 effector function
a) Immunoblot for phosphorylated and total AMPK and ACC in control and Opa1CD4Cre Th17 cells. ACTIN: sample processing control. Graph, relative signal intensity (n = 4, 2 independent experiments). b) IL-17A expression in control and Opa1CD4Cre CD4+ TN cells cultured in Th17 conditions and Compound C from day 0 (d0) or from day 3 to 4 (d3) and restimulated (n = 3). c) Left, immunoblot for AMPK and OPA1 in T cells transfected with Cas9 and indicated gRNAs and cultured 5 days in Th17 conditions. ACTIN: sample processing control. Right, IL-17A expression in restimulated cells (n = 3 biological and transfection replicates). d) Expression of phosphorylated S6 (n = 8) and 4E-BP1 (n = 7 biological and transfection replicates), 2 independent experiments. e) IL-17A expression in T cells transfected with Cas9 and indicated gRNAs and cultured in Th17 conditions 2 days followed by 48 h rapamycin treatment (n = 3 biological and transfection replicates). f) Phosphorylated H2Ax expression (n = 6, 2 independent experiments). g) Cyclic AMP (cAMP) levels (LC-MS, n = 8, 2 independent experiments). h) Basal calcium levels (n = 8, 2 independent experiments). i) Mitochondrial ROS (mitoSOX, n = 3). j) Control and Opa1CD4Cre CD4+ TN cells cultured in Th17 conditions 1 day followed by 2 day inhibitor-treatment. Graph, IL-17A MFI log2 FC inhibitor-treated vs. vehicle-treated cells (n = 7 biological replicates, except Tempol: n = 4 and NAC: n = 3, 2 independent experiments). Representative plots and graphs summarize results of at least two independent experiments, except noted otherwise. Data (a–i) are means ± s.e.m. Unpaired two-tailed t-test (a, f, g, h, i), two-sided Tukey’s test (b, c, d) or Šidák’s test (e, j). Exact P values are indicated.
