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. 2014 Oct 27;9(10):e104809. doi: 10.1371/journal.pone.0104809

Figure 3. TSC2 negatively regulates AdPLA2 expression in rapamycin-insensitive manner in vitro.

Figure 3

(A) Immunoblotting analysis of AdPLA2 and tuberin in TSC2-deficient (TSC2−) and TSCS2-addback (TSC2+) LAM patient-derived cells. Data show the mean of three sets of independent samples. Densitometry analysis of the protein levels of AdPLA2. (B) Secreted levels of prostaglandin E2 (PGE2) were quantified in conditioned media collected from TSC2-deficient (TSC2−) and TSC2-addback (TSC2+) LAM patient-derived cells using ELISA. Results are representative of three sets of independent samples per group. (C) Tsc2 −/− p53 −/− and Tsc2 +/+ p53 −/−MEFs were treated with 20 nM rapamycin for 24 hr. Levels of AdPLA2, tuberin and phospho-S6 (S235/236) were assessed by immunoblotting analysis. Results are representative of three different experiments. (D) Tsc2 −/− p53 −/− and Tsc2 +/+ p53 −/− MEFs were treated with 20 nM rapamycin (Rapa) or control for 24 hr. Secreted levels of 6-keto-PGF1α were quantified in conditioned media using ELISA. Results are representative of three sets of independent samples per group. (E) Rat-derived ELT3 cells were treated with 20 nM rapamycin (Rapa) or control for 24 hr. Immunoblotting analysis of AdPLA2 and tuberin were assessed. Results are representative of three different experiments. (F) Secreted levels of prostaglandin E2 (PGE2) were quantified in conditioned media collected from TSC2-deficient (TSC2−) and TSC2-addback (TSC2+) ELT3 cells using ELISA. Results are representative of three sets of independent samples per group. (G) Patient-derived TSC2-deficient (TSC2−) cells were treated with 20 nM rapamycin (Rapa), 100 nM Torin1, 50 µM PI-103, 50 µM PD98059 or control for 24 hr. Levels of AdPLA2, tuberin, phospho-Akt (S473) and phospho-Erk (T202/Y204) were assessed by immunoblotting analysis. *P<0.05, **P<0.01, Student’s t-test.