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Fig. 1. LPS simulates the <t>mTORC1</t> signaling in stable THP-1 macrophages. Stably transduced S,R,T THP-1 macrophages were treated with 1 μg LPS/ml media or PBS as control for 24 h. Macrophage cellular proteins were extracted in modified RIPA buffer and analyzed for Raptor, TSC2, phosphorylated p70S6K (Thr389), p70S6K, phosphorylated S6 (Ser235/236), S6 and β-actin by Western blotting. Relative band densitometry is graphed as box-and-whisker plots with reference to PBS control, n = 3 biological replicates. Statistical significance was assessed by the Student t-test; * P < 0.05 vs. PBS control.

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1) Product Images from "Divergent regulation of inflammatory cytokines by mTORC1 in THP-1-derived macrophages and intestinal epithelial Caco-2 cells."

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

Journal: Life sciences

doi: 10.1016/j.lfs.2021.119920

Fig. 1. LPS simulates the mTORC1 signaling in stable THP-1 macrophages. Stably transduced S,R,T THP-1 macrophages were treated with 1 μg LPS/ml media or PBS as control for 24 h. Macrophage cellular proteins were extracted in modified RIPA buffer and analyzed for Raptor, TSC2, phosphorylated p70S6K (Thr389), p70S6K, phosphorylated S6 (Ser235/236), S6 and β-actin by Western blotting. Relative band densitometry is graphed as box-and-whisker plots with reference to PBS control, n = 3 biological replicates. Statistical significance was assessed by the Student t-test; * P < 0.05 vs. PBS control.

Figure Legend Snippet: Fig. 1. LPS simulates the mTORC1 signaling in stable THP-1 macrophages. Stably transduced S,R,T THP-1 macrophages were treated with 1 μg LPS/ml media or PBS as control for 24 h. Macrophage cellular proteins were extracted in modified RIPA buffer and analyzed for Raptor, TSC2, phosphorylated p70S6K (Thr389), p70S6K, phosphorylated S6 (Ser235/236), S6 and β-actin by Western blotting. Relative band densitometry is graphed as box-and-whisker plots with reference to PBS control, n = 3 biological replicates. Statistical significance was assessed by the Student t-test; * P < 0.05 vs. PBS control.

Techniques Used: Stable Transfection, Control, Modification, Western Blot, Whisker Assay

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

Fig. 5. mTORC1 asymmetrically regulates cytokine gene expression in THP-1 macrophages and Caco-2 cells exposed to a pro-inflammatory environment. a) Stable S,R,T THP-1 macrophages were incubated with 1 μg LPS/ml media or PBS as control for 24 h. b) Stable S,R,T Caco-2 cells were incubated for 24 h with the CM of THP-1 shTSC2 macrophages treated with PBS or 1 μg LPS/ml media. Non-linear regression analysis was performed to correlate cytokine (IL- 6, IL-8, TNFα, IL-10) gene expression with mTORC1 activity. * indicates a significant effect of mTORC1 activity level on cytokine gene expression (one- factor ANOVA, P < 0.05, n = 3).

Figure Legend Snippet: Fig. 5. mTORC1 asymmetrically regulates cytokine gene expression in THP-1 macrophages and Caco-2 cells exposed to a pro-inflammatory environment. a) Stable S,R,T THP-1 macrophages were incubated with 1 μg LPS/ml media or PBS as control for 24 h. b) Stable S,R,T Caco-2 cells were incubated for 24 h with the CM of THP-1 shTSC2 macrophages treated with PBS or 1 μg LPS/ml media. Non-linear regression analysis was performed to correlate cytokine (IL- 6, IL-8, TNFα, IL-10) gene expression with mTORC1 activity. * indicates a significant effect of mTORC1 activity level on cytokine gene expression (one- factor ANOVA, P < 0.05, n = 3).

Techniques Used: Gene Expression, Incubation, Control, Activity Assay

Fig. 6. Schematic diagrams of AKT, ERK and NFκB signaling in S,R,T Caco-2 cells exposed to the conditioned media of THP-1-derived macrophages treated with LPS. It is proposed that, in Caco-2 cells exposed to this pro- inflammatory environment, the mTORC1/IRS/PI3K/AKT signaling plays an important role in mediating ERK and NFκB-dependent expression of cytokines. a) Growth factors and cytokines found in the conditioned media of THP-1- derived macrophages activate the Ras/MAPK and PI3K/AKT pathways and various interleukin receptors in Caco-2 cells to induce ERK and NFκB-mediated expression of cytokines. Depending on Caco-2 cells' mTORC1 activity level, the IRS/PI3K/AKT pathway can upregulate or downregulate ERK and NFκB-medi ated expression of cytokines on the basis that (i) PI3K interacts with Ras [70], (ii) the PI3K inhibitor LY294002 represses both AKT and ERK signaling in Caco- 2 cells whereas MEK1/2 inhibitor UO126 only represses ERK signaling (Fig. S3), (iii) AKT can stimulate IKK [71], and (iv) elevated mTORC1 activity downregulates the IRS/PI3K/AKT pathway via inhibitory phosphorylation of IRS by p70S6K [32]. b) mTORC1 suppression, such as through the knockdown of Raptor, can coincide with NFκB activation since mTORC1 depletion eases the negative feedback of p70S6K on the IRS/PI3K/AKT pathway thereby stimu lating the Ras/ERK and IKK/NFκB signaling pathways in a PI3K/AKT- dependent manner. c) In contrast, under persistent mTORC1 activation, such as through the knockdown of TSC2, the IRS/PI3K/AKT pathway is repressed and in turn ERK and IKK-mediated expression of cytokines is decreased. Of note, the contributions of interleukins 1β, 6 and 8 to the response of Caco-2 cells is contingent upon the expression levels of their respective receptors, which can be low in regular Caco-2 cells, suggesting that other cytokines and chemokines produced by THP-1 macrophages may be at play.

Figure Legend Snippet: Fig. 6. Schematic diagrams of AKT, ERK and NFκB signaling in S,R,T Caco-2 cells exposed to the conditioned media of THP-1-derived macrophages treated with LPS. It is proposed that, in Caco-2 cells exposed to this pro- inflammatory environment, the mTORC1/IRS/PI3K/AKT signaling plays an important role in mediating ERK and NFκB-dependent expression of cytokines. a) Growth factors and cytokines found in the conditioned media of THP-1- derived macrophages activate the Ras/MAPK and PI3K/AKT pathways and various interleukin receptors in Caco-2 cells to induce ERK and NFκB-mediated expression of cytokines. Depending on Caco-2 cells' mTORC1 activity level, the IRS/PI3K/AKT pathway can upregulate or downregulate ERK and NFκB-medi ated expression of cytokines on the basis that (i) PI3K interacts with Ras [70], (ii) the PI3K inhibitor LY294002 represses both AKT and ERK signaling in Caco- 2 cells whereas MEK1/2 inhibitor UO126 only represses ERK signaling (Fig. S3), (iii) AKT can stimulate IKK [71], and (iv) elevated mTORC1 activity downregulates the IRS/PI3K/AKT pathway via inhibitory phosphorylation of IRS by p70S6K [32]. b) mTORC1 suppression, such as through the knockdown of Raptor, can coincide with NFκB activation since mTORC1 depletion eases the negative feedback of p70S6K on the IRS/PI3K/AKT pathway thereby stimu lating the Ras/ERK and IKK/NFκB signaling pathways in a PI3K/AKT- dependent manner. c) In contrast, under persistent mTORC1 activation, such as through the knockdown of TSC2, the IRS/PI3K/AKT pathway is repressed and in turn ERK and IKK-mediated expression of cytokines is decreased. Of note, the contributions of interleukins 1β, 6 and 8 to the response of Caco-2 cells is contingent upon the expression levels of their respective receptors, which can be low in regular Caco-2 cells, suggesting that other cytokines and chemokines produced by THP-1 macrophages may be at play.

Techniques Used: Derivative Assay, Expressing, Activity Assay, Phospho-proteomics, Knockdown, Activation Assay, Protein-Protein interactions, Produced

Image Search Results

Simvastatin administration during fasting activates SREBP-2 and autophagy and augments FFA-induced APOA1 expression. (A) Huh7 cells cultured under serum-depleted conditions were treated with simvastatin (1, 5, or 10 μM) for 24 h. APOA1, PLTP, and GK mRNA levels were quantified by qPCR, normalized to the geometric mean of HPRT and B2M , and expressed as fold change relative to untreated controls. (B) Simvastatin (10 μM) enhances sodium oleate (50 μM)–induced APOA1 expression in serum-depleted Huh7 cells. Gene expression was measured by qPCR and normalized to HPRT and B2M . (C) Fasting-phase simvastatin treatment upregulates hepatic Srebf2 and its target genes in vivo . A/J mice received simvastatin by gavage for 5 weeks and were sacrificed 5 h after the final dose (ZT09); hepatic mRNA levels were normalized to Hprt . (D) Expression of autophagy-related genes ( Atg7 , Atg12 , Becn1 and others) in liver following fasting-phase simvastatin treatment. (E) Pharmacologic inhibition of autophagy attenuates simvastatin-induced APOA1 expression in Huh7 cells. Serum-starved cells were treated with simvastatin in the presence or absence of 500 µM leucine (mTORC1 activator) or 50 µM leupeptin (lysosomal protease inhibitor); APOA1 mRNA was measured by qPCR and normalized to HPRT1 and B2M . Data are mean ± SEM with individual data points overlaid (n indicated in each panel). Statistical significance: p < 0.05; * p < 0.01; ** p < 0.001 (two-way ANOVA with Tukey’s post hoc test).

Journal: Frontiers in Pharmacology

Article Title: TLR4 modulates simvastatin’s impact on HDL cholesterol and glycemic control

doi: 10.3389/fphar.2025.1655873

Figure Lengend Snippet: Simvastatin administration during fasting activates SREBP-2 and autophagy and augments FFA-induced APOA1 expression. (A) Huh7 cells cultured under serum-depleted conditions were treated with simvastatin (1, 5, or 10 μM) for 24 h. APOA1, PLTP, and GK mRNA levels were quantified by qPCR, normalized to the geometric mean of HPRT and B2M , and expressed as fold change relative to untreated controls. (B) Simvastatin (10 μM) enhances sodium oleate (50 μM)–induced APOA1 expression in serum-depleted Huh7 cells. Gene expression was measured by qPCR and normalized to HPRT and B2M . (C) Fasting-phase simvastatin treatment upregulates hepatic Srebf2 and its target genes in vivo . A/J mice received simvastatin by gavage for 5 weeks and were sacrificed 5 h after the final dose (ZT09); hepatic mRNA levels were normalized to Hprt . (D) Expression of autophagy-related genes ( Atg7 , Atg12 , Becn1 and others) in liver following fasting-phase simvastatin treatment. (E) Pharmacologic inhibition of autophagy attenuates simvastatin-induced APOA1 expression in Huh7 cells. Serum-starved cells were treated with simvastatin in the presence or absence of 500 µM leucine (mTORC1 activator) or 50 µM leupeptin (lysosomal protease inhibitor); APOA1 mRNA was measured by qPCR and normalized to HPRT1 and B2M . Data are mean ± SEM with individual data points overlaid (n indicated in each panel). Statistical significance: p < 0.05; * p < 0.01; ** p < 0.001 (two-way ANOVA with Tukey’s post hoc test).

Article Snippet: For fasting-mimetic conditions ( ; ), cells were washed twice with 1× PBS, incubated overnight in serum-free DMEM, and then treated for 24 h with 5 μM simvastatin alone or in combination with one or more of the following: 50 μM oleic acid (MilliporeSigma; C18:1, #O1383, purity >99% by GC); 10 μM GW6471 (MedChemExpress; #HY-15372, purity 99%), a selective PPARα antagonist; 500 μM leucine (MedChemExpress; #HY-N0486, purity 98%), which suppresses autophagy initiation via mTORC1 activation; or 50 μM leupeptin (MedChemExpress; #HY-18234A, purity 99.39%), a cysteine/serine/threonine protease inhibitor that blocks autophagic flux ( ; ; ).

Techniques: Expressing, Cell Culture, Gene Expression, In Vivo, Inhibition, Protease Inhibitor

Role of mammalian target of rapamycin complex 1 (mTORC1) and PGC1α in propionate-mediated regulation of the expression of gluconeogenesis-related genes in calf hepatocytes. (A–C). Calf hepatocytes were treated with NaP and rapamycin (100 nM). The expression levels of FBP1 (A), PCK1 (B), and G6PC (C) were detected by RT-qPCR. (D–F). Calf hepatocytes were treated with NaP and MHY1485 (2 μM). The expression levels of FBP1 (D), PCK1 (E), and G6PC (F) were detected by RT-qPCR. (G–I). Calf hepatocytes were treated with NaP and SR18292 (20 μM). The expression levels of FBP1 (G), PCK1 (H), and G6PC (I) were detected by RT-qPCR. Data were analyzed by two-way ANOVA. a, b, c Bars with a different letter mean a significant difference ( P < 0.05).

Journal: Animal Nutrition

Article Title: Propionate promotes gluconeogenesis by regulating mechanistic target of rapamycin (mTOR) pathway in calf hepatocytes

doi: 10.1016/j.aninu.2023.07.001

Figure Lengend Snippet: Role of mammalian target of rapamycin complex 1 (mTORC1) and PGC1α in propionate-mediated regulation of the expression of gluconeogenesis-related genes in calf hepatocytes. (A–C). Calf hepatocytes were treated with NaP and rapamycin (100 nM). The expression levels of FBP1 (A), PCK1 (B), and G6PC (C) were detected by RT-qPCR. (D–F). Calf hepatocytes were treated with NaP and MHY1485 (2 μM). The expression levels of FBP1 (D), PCK1 (E), and G6PC (F) were detected by RT-qPCR. (G–I). Calf hepatocytes were treated with NaP and SR18292 (20 μM). The expression levels of FBP1 (G), PCK1 (H), and G6PC (I) were detected by RT-qPCR. Data were analyzed by two-way ANOVA. a, b, c Bars with a different letter mean a significant difference ( P < 0.05).

Article Snippet: Cells were maintained in RPMI 1640 basic medium containing 2% BSA and treated with different concentrations of PA (0, 100, 200, or 400 μM) and NaP (0, 1, 2.5, or 5 mM), alone or in combination, for 12 h. A 2 × 2 factorial arrangement was applied for the experiments: primary hepatocytes were treated with NaP (2.5 mM), the mTORC1 inhibitor rapamycin (100 nM) (V900930; Sigma Aldrich, MO, USA), the mTORC1 activator MHY1485 (2 μM) (S7811; Selleck, Shanghai, China), and the PGC1α inhibitor SR-18292 (20 μM) (S8528; Selleck) for 12 h to observe the effect of mTORC1 and PGC1α on the mRNA expression of gluconeogenic genes.

Techniques: Expressing, Quantitative RT-PCR

Immunohistochemical analysis of mTOR and metabolic pathway markers in papillary (PRCC) and clear cell renal cell carcinomas (CCRCC). The expression of p-mTOR (marker for mTOR kinase activity), p-S6 (mTORC1 activity marker), Rictor (marker of the amount of mTORC2), GLUT1, HXK2, PFKP (glycolysis markers), G6PD (pentose phosphate pathway marker), GLS (glutaminolysis marker), ACSS2 (marker for acetate consumption), CPT1A (fatty acid β-oxidation marker), ATPB (OXPHOS marker), COX IV, and TOM20 (mitochondrial markers) was analyzed in normal kidney, PRCCs, and CCRCCs. The detected characteristic differences in enzyme expression profiles between PRCCs and CCRCCs were highlighted by red frame. Immunohistochemistry (DAB chromogen–brown) and hematoxylin counterstaining were used. Scale bars indicate 50 μm.

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics

doi: 10.3390/ijms231810587

Figure Lengend Snippet: Immunohistochemical analysis of mTOR and metabolic pathway markers in papillary (PRCC) and clear cell renal cell carcinomas (CCRCC). The expression of p-mTOR (marker for mTOR kinase activity), p-S6 (mTORC1 activity marker), Rictor (marker of the amount of mTORC2), GLUT1, HXK2, PFKP (glycolysis markers), G6PD (pentose phosphate pathway marker), GLS (glutaminolysis marker), ACSS2 (marker for acetate consumption), CPT1A (fatty acid β-oxidation marker), ATPB (OXPHOS marker), COX IV, and TOM20 (mitochondrial markers) was analyzed in normal kidney, PRCCs, and CCRCCs. The detected characteristic differences in enzyme expression profiles between PRCCs and CCRCCs were highlighted by red frame. Immunohistochemistry (DAB chromogen–brown) and hematoxylin counterstaining were used. Scale bars indicate 50 μm.

Article Snippet: 32 , 0.1111111 , – , #2211 , Cell Signaling , mTORC1 activity , Anti-phospho(Ser235/236)-S6.

Techniques: Immunohistochemical staining, Expressing, Marker, Activity Assay, Immunohistochemistry

LC–MS analyses of metabolic features and inhibitor sensitivity of papillary (ACHN) and clear cell renal cell carcinoma (786-O) cell lines. ( A ) LC–MS analysis of the intracellular metabolite concentrations in papillary (ACHN), clear cell (786-O) RCC and normal tubular epithelial (HK-2) cell lines. Lactate/pyruvate, pyruvate/citrate, and lactate/malate ratios were used to assess the glycolytic capacity and the activity of the TCA cycle of the cell lines (CIT–citrate, iCIT–isocitrate, α-KG–alpha-ketoglutarate, s-CoA–succinyl-CoA, SUC–succinate, FUM–fumarate, MAL–malate, OA–oxaloacetate), whereas lactate/glutamate ratio was used to evaluate the glutamine utilization. ( B ) The mTORC1 (rapamycin–rapa) and metabolic inhibitors (ACSS2i–inhibitor of acetate utilization, BPTES–glutaminolysis inhibitor, metformin–metf–AMPK-inhibitor, doxycycline–doxy–antibiotics with mitochondrial inhibitory effect) sensitivity of papillary (ACHN) and clear cell (786-O) RCC cell lines were evaluated as monotherapy or in combination. Regarding the 786-O cells, the treatments were performed both in RPMI-1640 (2000 mg/L glucose) medium under generally applied and optimized conditions and in DMEM high glucose (4500 mg/L)–to compare the role of maintaining parameters with similar glucose concentration levels as ACHN and HK-2 cells. * p values below 0.05, ** p values below 0.01 (one-way ANOVA with Tukey’s post hoc test), and at least 20% decrease in proliferation were considered biologically relevant. Synergistic treatment interactions were labeled with S (based on combination index calculation).

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics

doi: 10.3390/ijms231810587

Figure Lengend Snippet: LC–MS analyses of metabolic features and inhibitor sensitivity of papillary (ACHN) and clear cell renal cell carcinoma (786-O) cell lines. ( A ) LC–MS analysis of the intracellular metabolite concentrations in papillary (ACHN), clear cell (786-O) RCC and normal tubular epithelial (HK-2) cell lines. Lactate/pyruvate, pyruvate/citrate, and lactate/malate ratios were used to assess the glycolytic capacity and the activity of the TCA cycle of the cell lines (CIT–citrate, iCIT–isocitrate, α-KG–alpha-ketoglutarate, s-CoA–succinyl-CoA, SUC–succinate, FUM–fumarate, MAL–malate, OA–oxaloacetate), whereas lactate/glutamate ratio was used to evaluate the glutamine utilization. ( B ) The mTORC1 (rapamycin–rapa) and metabolic inhibitors (ACSS2i–inhibitor of acetate utilization, BPTES–glutaminolysis inhibitor, metformin–metf–AMPK-inhibitor, doxycycline–doxy–antibiotics with mitochondrial inhibitory effect) sensitivity of papillary (ACHN) and clear cell (786-O) RCC cell lines were evaluated as monotherapy or in combination. Regarding the 786-O cells, the treatments were performed both in RPMI-1640 (2000 mg/L glucose) medium under generally applied and optimized conditions and in DMEM high glucose (4500 mg/L)–to compare the role of maintaining parameters with similar glucose concentration levels as ACHN and HK-2 cells. * p values below 0.05, ** p values below 0.01 (one-way ANOVA with Tukey’s post hoc test), and at least 20% decrease in proliferation were considered biologically relevant. Synergistic treatment interactions were labeled with S (based on combination index calculation).

Article Snippet: 32 , 0.1111111 , – , #2211 , Cell Signaling , mTORC1 activity , Anti-phospho(Ser235/236)-S6.

Techniques: Liquid Chromatography with Mass Spectroscopy, Activity Assay, Concentration Assay, Labeling

Antibodies used for Protein Expression Analyses.

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics

doi: 10.3390/ijms231810587

Figure Lengend Snippet: Antibodies used for Protein Expression Analyses.

Article Snippet: 32 , 0.1111111 , – , #2211 , Cell Signaling , mTORC1 activity , Anti-phospho(Ser235/236)-S6.

Techniques: Expressing, Activity Assay, Phospho-proteomics, Marker, Control

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. 1. LPS simulates the mTORC1 signaling in stable THP-1 macrophages. Stably transduced S,R,T THP-1 macrophages were treated with 1 μg LPS/ml media or PBS as control for 24 h. Macrophage cellular proteins were extracted in modified RIPA buffer and analyzed for Raptor, TSC2, phosphorylated p70S6K (Thr389), p70S6K, phosphorylated S6 (Ser235/236), S6 and β-actin by Western blotting. Relative band densitometry is graphed as box-and-whisker plots with reference to PBS control, n = 3 biological replicates. Statistical significance was assessed by the Student t-test; * P < 0.05 vs. PBS control.

Article Snippet: Stable lines of THP-1 monocytes and Caco-2 epithelial cells in which mTORC1 activity was either (i) upregulated by knocking down TSC2, cell line referred to as shTSC2 or T, (ii) downregulated by knocking down Raptor, cell line referred to as shRaptor or R, or (iii) at baseline by using a scramble shRNA sequence resulting in unchanged mTORC1 activity, cell line referred to as shScramble or S were generated by using lentiviral shRNA. pLKO.1-TSC2 (Addgene plasmid #15478) was from Do-Hyung Kim [49]. pLKO.1-Raptor (Addgene plasmid #1858) and pLKO.1 scramble shRNA (Addgene plasmid #1864) were from David Sabatini [50].

Techniques: Stable Transfection, Control, Modification, Western Blot, Whisker Assay

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.

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.

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.

Techniques: Gene Expression, Quantitative RT-PCR, Cell Culture, 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. 5. mTORC1 asymmetrically regulates cytokine gene expression in THP-1 macrophages and Caco-2 cells exposed to a pro-inflammatory environment. a) Stable S,R,T THP-1 macrophages were incubated with 1 μg LPS/ml media or PBS as control for 24 h. b) Stable S,R,T Caco-2 cells were incubated for 24 h with the CM of THP-1 shTSC2 macrophages treated with PBS or 1 μg LPS/ml media. Non-linear regression analysis was performed to correlate cytokine (IL- 6, IL-8, TNFα, IL-10) gene expression with mTORC1 activity. * indicates a significant effect of mTORC1 activity level on cytokine gene expression (one- factor ANOVA, P < 0.05, n = 3).

Techniques: Gene Expression, Incubation, Control, Activity Assay

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. 6. Schematic diagrams of AKT, ERK and NFκB signaling in S,R,T Caco-2 cells exposed to the conditioned media of THP-1-derived macrophages treated with LPS. It is proposed that, in Caco-2 cells exposed to this pro- inflammatory environment, the mTORC1/IRS/PI3K/AKT signaling plays an important role in mediating ERK and NFκB-dependent expression of cytokines. a) Growth factors and cytokines found in the conditioned media of THP-1- derived macrophages activate the Ras/MAPK and PI3K/AKT pathways and various interleukin receptors in Caco-2 cells to induce ERK and NFκB-mediated expression of cytokines. Depending on Caco-2 cells' mTORC1 activity level, the IRS/PI3K/AKT pathway can upregulate or downregulate ERK and NFκB-medi ated expression of cytokines on the basis that (i) PI3K interacts with Ras [70], (ii) the PI3K inhibitor LY294002 represses both AKT and ERK signaling in Caco- 2 cells whereas MEK1/2 inhibitor UO126 only represses ERK signaling (Fig. S3), (iii) AKT can stimulate IKK [71], and (iv) elevated mTORC1 activity downregulates the IRS/PI3K/AKT pathway via inhibitory phosphorylation of IRS by p70S6K [32]. b) mTORC1 suppression, such as through the knockdown of Raptor, can coincide with NFκB activation since mTORC1 depletion eases the negative feedback of p70S6K on the IRS/PI3K/AKT pathway thereby stimu lating the Ras/ERK and IKK/NFκB signaling pathways in a PI3K/AKT- dependent manner. c) In contrast, under persistent mTORC1 activation, such as through the knockdown of TSC2, the IRS/PI3K/AKT pathway is repressed and in turn ERK and IKK-mediated expression of cytokines is decreased. Of note, the contributions of interleukins 1β, 6 and 8 to the response of Caco-2 cells is contingent upon the expression levels of their respective receptors, which can be low in regular Caco-2 cells, suggesting that other cytokines and chemokines produced by THP-1 macrophages may be at play.

Techniques: Derivative Assay, Expressing, Activity Assay, Phospho-proteomics, Knockdown, Activation Assay, Protein-Protein interactions, Produced

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