Journal: Life sciences

Article Title: Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells.

doi: 10.1016/j.lfs.2021.119920

Figure Lengend Snippet: Fig. 2. mTORC1 potentiates the LPS-mediated inflammatory response of THP-1 macrophages. a) S,R,T THP-1 macrophages were stimulated with 1 μg LPS/ml media or PBS as control for 24 h. After 24 h, conditioned media were collected, and cellular total RNA was extracted. Cytokine gene expression (TNFα, IL-6, IL- 8, IL-1β and IL-10) was measured by qRT-PCR and normalized to housekeeping gene, PPIA. Media IL-6 and IL-8 levels were measured by ELISA. Values not sharing a common letter (a,b,c,d,e next to the horizontal bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05, n = 3–6. b) Validation of LYS6K2 efficacy (an inhibitor of p70S6K, 10 μM in DMF) as determined by a 66% decrease of S6 (Ser235/236) phosphorylation in THP-1 shScramble macrophages. Statistical significance was assessed by the Student t-test; * P < 0.05 vs. DMF control, n = 3. c) LYS6K2 (10 μM) abrogated LPS-mediated IL-6 gene expression in THP-1 shScramble macrophages. Values not sharing a common letter (a,b,c next to the horizontal bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05, n = 3.

Article Snippet: Total RNA was isolated from cells using Aurum Total RNA Mini Kit (BioRad) that includes a DNase I incubation step.

Techniques: Control, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Phospho-proteomics

Journal: Life sciences

Article Title: Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells.

doi: 10.1016/j.lfs.2021.119920

Figure Lengend Snippet: Fig. 3. Conditioned media (CM) from LPS-treated THP-1 macrophages activate mTORC1 and cytokine gene expression in Caco-2 cells. a) Stably transduced THP-1 macrophages were stimulated with 1 μg LPS/ml media or PBS as control for 24 h, the CM were collected and transferred to 80% confluent regular Caco-2 cells. Caco-2 cells were cultured in the presence of THP-1 CM for 24 h. A treatment group of Caco-2 cells cultured in fresh THP-1 medium for 24 h was used as a negative control. To control for possible LPS carry over in THP-1 CM, a separate group of Caco-2 cells were cultured for 24 h in CM from THP-1 shScramble macrophages treated with PBS to which fresh LPS (1 μg LPS/ml media) was added. After 24 h, total RNA preparations of Caco-2 cells were analyzed for changes in cytokine gene expression (IL- 6, IL-8, IL-10) by qRT-PCR and normalized to housekeeping gene, PPIA. Values not sharing a common letter (a,b,c next to the horizontal bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05, n = 3–6. b) Separate groups of 80% confluent regular Caco-2 cells were cultured for 24 h in CM from PBS or LPS-treated THP-1 shTSC2 macrophages. After 24 h, cellular proteins were extracted in modified RIPA buffer and analyzed for phos phorylated p70S6K (Thr389), p70S6K, phosphorylated p85S6K (Thr412), p85S6K, phosphorylated S6 (Ser235/236), S6, phosphorylated ERK (Thr202/Tyr204), ERK, COX-2, and β-actin by Western blotting. Statistical significance was assessed by the Student t-test; * P < 0.05, n = 3, ns = not significant.

Article Snippet: Total RNA was isolated from cells using Aurum Total RNA Mini Kit (BioRad) that includes a DNase I incubation step.

Techniques: Gene Expression, Stable Transfection, Control, Cell Culture, Negative Control, Quantitative RT-PCR, Modification, Western Blot

Journal: Life sciences

Article Title: Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells.

doi: 10.1016/j.lfs.2021.119920

Figure Lengend Snippet: Fig. 4. mTORC1 protects Caco-2 cells exposed to secretions of LPS-treated THP-1 shTSC2 cells. a) Stable S,R,T Caco-2 cells received the CM of PBS or LPS-treated THP-1 shTSC2 macrophages for 24 h. After 24 h, total RNA preparations were analyzed for cytokine (IL-6, IL-8, TNFα, IL-10) gene expression by qRT-PCR and normalized to housekeeping gene, PPIA, n = 3. b) Caco-2 shTSC2 cells were pre-treated with rapamycin (200 nM) for 1 h, then media was replaced with the CM from THP-1 shTSC2 treated +/− LPS and supplemented with rapamycin (200 nM). After 24 h, total RNA preparations were analyzed for cytokine (TNFα, IL-10) gene expression by qRT-PCR and normalized to housekeeping gene, PPIA, n = 3. c) Stable S,R,T Caco-2 cells were cultured in complete EMEM media until 80% confluence, at which point cellular proteins were extracted in RIPA buffer and analyzed for p-ERK (Thr202/Tyr204), ERK, p-AKT (Ser473), and AKT by Western blotting, n = 3. a-c) Values not sharing a common letter (a,b,c,d next to the bars) are significantly different. One-way ANOVA followed by Tukey's multiple comparisons test; P < 0.05.

Article Snippet: Total RNA was isolated from cells using Aurum Total RNA Mini Kit (BioRad) that includes a DNase I incubation step.

Techniques: Gene Expression, Quantitative RT-PCR, Cell Culture, Western Blot