Figure 2. TSC2 negatively regulates COX-2 expression and prostaglandin production in a rapamycin-insensitive but Torin 1 and mTORC2-dependent manner in vitro. (a) Simplified prostaglandin biosynthesis pathway. (b) Re-analysis of previously published expression array data (Lee et al., 2010). Bar graph of the transcript levels of PTGS2 (COX-2) and PTGIS (prostacyclin synthase) in TSC2-deficient (TSC−) and TSCS2-addback (TSC2+) LAM patient–derived cells treated with rapamycin (Rapa) or Vehicle for 24 h. Data show the mean of three sets of independent samples. (c) Real-time RT-PCR analysis of the transcript levels of PTGS2 and PTGIS in TSC2-deficient LAM patient–derived cells relative to TSC2-addback cells treated with 20 nM rapamycin or vehicle for 24 h. Data show the mean of three sets of independent samples. (d–f) Immunoblot analyses of tuberin, PTGIS, phospho-S6 (S235/236), and COX-2 protein in LAM patient–derived cell treated with 20 nM rapamycin (Rapa), 1 µg/ml tunicamycin (TN), or rapamycin plus tunicamycin, for 24 h. Results are representative of three different experiments. (e and g) Secreted prostaglandin levels were quantified in conditioned media collected from LAM patient–derived cells treated with 20 nM rapamycin (Rapa), 1 µg/ml tunicamycin (TN), or rapamycin plus tunicamycin, for 24 h (n = 3; ELISA). Results are representative of three sets of independent samples per group. (h) ELT3 cells were treated with 20 nM rapamycin (Rapa) or control for 24 h. Immunoblot analysis of COX-2 and phospho-S6 (S235/236). Results are representative of three different experiments. (i) Secreted prostaglandin levels were quantified in conditioned media collected from ELT3 cells treated with rapamycin or control. Data were normalized to control group (n = 3; ELISA). Results are representative of three sets of independent samples per group. (j) HeLa, OVCAR-5, and U2OS cells were treated with 20 nM rapamycin for 24 h. Immunoblot analysis of COX-2 and phospho-S6 (S235/236). Results are representative of three different experiments. (k) Secreted prostaglandin levels were quantified in conditioned media collected from cells treated with rapamycin or control, data were normalized to control group (n = 3; ELISA). Results are representative of three sets of independent samples per group. *, P < 0.05; **, P < 0.01; ***, P < 0.005; Student’s t test.
Figure 2.

TSC2 negatively regulates COX-2 expression and prostaglandin production in a rapamycin-insensitive but Torin 1 and mTORC2-dependent manner in vitro. (a) Simplified prostaglandin biosynthesis pathway. (b) Re-analysis of previously published expression array data (Lee et al., 2010). Bar graph of the transcript levels of PTGS2 (COX-2) and PTGIS (prostacyclin synthase) in TSC2-deficient (TSC−) and TSCS2-addback (TSC2+) LAM patient–derived cells treated with rapamycin (Rapa) or Vehicle for 24 h. Data show the mean of three sets of independent samples. (c) Real-time RT-PCR analysis of the transcript levels of PTGS2 and PTGIS in TSC2-deficient LAM patient–derived cells relative to TSC2-addback cells treated with 20 nM rapamycin or vehicle for 24 h. Data show the mean of three sets of independent samples. (d–f) Immunoblot analyses of tuberin, PTGIS, phospho-S6 (S235/236), and COX-2 protein in LAM patient–derived cell treated with 20 nM rapamycin (Rapa), 1 µg/ml tunicamycin (TN), or rapamycin plus tunicamycin, for 24 h. Results are representative of three different experiments. (e and g) Secreted prostaglandin levels were quantified in conditioned media collected from LAM patient–derived cells treated with 20 nM rapamycin (Rapa), 1 µg/ml tunicamycin (TN), or rapamycin plus tunicamycin, for 24 h (n = 3; ELISA). Results are representative of three sets of independent samples per group. (h) ELT3 cells were treated with 20 nM rapamycin (Rapa) or control for 24 h. Immunoblot analysis of COX-2 and phospho-S6 (S235/236). Results are representative of three different experiments. (i) Secreted prostaglandin levels were quantified in conditioned media collected from ELT3 cells treated with rapamycin or control. Data were normalized to control group (n = 3; ELISA). Results are representative of three sets of independent samples per group. (j) HeLa, OVCAR-5, and U2OS cells were treated with 20 nM rapamycin for 24 h. Immunoblot analysis of COX-2 and phospho-S6 (S235/236). Results are representative of three different experiments. (k) Secreted prostaglandin levels were quantified in conditioned media collected from cells treated with rapamycin or control, data were normalized to control group (n = 3; ELISA). Results are representative of three sets of independent samples per group. *, P < 0.05; **, P < 0.01; ***, P < 0.005; Student’s t test.

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