mTORC2 regulates COX-2 expression via PI3K–AKT pathway in TSC2-deficient cells. (a) Tsc2^−/−p53^−/− MEFs were treated with 20 nM rapamycin, 250 nM Torin 1, or 20 µM LY294002 for 24 h. Levels of COX-2, 4EBP1, and phospho-S6 (S235/236) were assessed by immunoblot. (b and c) Tsc2^−/−p53^−/− MEFs were transfected with Rictor shRNA or control shRNA, and then selected with puromycin to obtain stable cells. Control shRNA or with Rictor shRNA-Tsc2^−/−p53^−/− MEFs were treated with vehicle or 20 nM rapamycin for 24 h. Levels of COX-2, COX-1, phospho-S6 (S235/236), and phospho-Akt (S473) were assessed by immunoblot. (d) LAM patient–derived cells (TSC⁻) cells were treated with 20 nM rapamycin, or 250 nM Torin 1, or 50 µM NS398 for 24 h. Levels of COX-2 and phospho-S6 (S235/236) were assessed by immunoblot. (e) TSC2-deficient LAM patient–derived cells were treated with 250 nM Torin 1 for 24 h. Levels of COX-2, phospho-p44/42–MAPK, phospho-Akt S473, and phospho-S6 (S235/236) were assessed by immunoblot. (f) TSC2-deficient LAM patient–derived cells were treated with 5 µM PI-103 or 5 µM AKTVIII for 24 h. Levels of COX-2 and phospho-Akt (S473) were assessed by immunoblot. (g) TSC2-deficient LAM patient–derived cells were treated with 50 µM PD98059, 5 µM PI-103, or PD98059 plus PI-103 for 24 h. Levels of COX-2, phospho-Akt S473, phospho-p44/42–MAPK, and phospho-S6 (S235/236) were assessed by immunoblot. (h) TSC2-deficient LAM patient–derived cells were treated with 1 µM Gefitinib or 1 µM Afatinib for 24 h. Levels of COX-2, EGFR, and phospho-Akt S473 were assessed by immunoblot. (i) ELT3 cells expressing empty vector (TSC⁻) and TSC2-addback (TSC2⁺), or LAM patient–derived cells (TSC⁻) cells and TSC2-addback cells (TSC2⁺) were treated with 20 nM rapamycin (Rapa) for 24 h. Subcellular localization of EGFR was examined using confocal microscopy. Bar, 10 µM. (j) Cells were treated with 50 µM Sulindac, 50 µM NS398, or 450 µM aspirin for 24 h. Levels of COX-1, COX-2, phospho-p44/42 MAPK, and phospho-S6 were assessed by immunoblot. (a–j) Results are representative of two to four different experiments. (k) PGE₂ levels from conditioned media were measured (ELISA). Results are representative of six sets of independent samples per group. (l) Cell proliferation after drug treatment was measured using MTT assay. Results are representative of 12 sets of independent samples per group. *, P < 0.05; Student’s t test.