mtorc1 inhibitor torin 1  (Tocris)

Journal: Reproduction (Cambridge, England)

Article Title: Manipulating bovine granulosa cell energy metabolism limits inflammation

doi: 10.1530/REP-20-0554

Figure Lengend Snippet: AICAR limits LPS-stimulated inflammation in granulosa cells. Granulosa cells from 4–8 mm or > 8.5 mm diameter ovarian follicles were treated in culture medium with vehicle, or for the indicated times with 1 mM AICAR (A), or for 24 h with 1 μg/mL LPS, 1 mM 2-deoxy-D-glucose (2-DG), 1 mM AICAR, 500 nM rapamycin or 50 nM Torin 1 (B), and the phosphorylation of acetyl-CoA carboxylase (ACC) analysed by Western blot. Representative Western blots of phosphorylated ACC, total ACC, and β-actin or α-tubulin are shown (left panel; Supplementary Fig. 1); with quantification of band densities for phosphorylated relative to total ACC (right panel). Data from independent experiments are presented as dot plots, with bars indicating the mean (S.E.M.). Data were analysed by ANOVA, with Dunnett’s post hoc test, and P-values reported. Granulosa cells from 4–8 mm (C, D, E and F) or > 8.5 mm diameter follicles (G, H, I and J) were treated for 2 h with culture medium containing vehicle (V) or the indicated final concentrations of AICAR, followed by a 24 h challenge with control medium or medium containing 1 μg/mL LPS, in the presence of the treatments. Supernatant IL-1α (C, G), IL-1β (D, H) and IL-8 (E, I) were measured by ELISA. Cell viability was assessed by MTT assay (F, J). Data from independent experiments are presented as dot plots, with bars indicating the mean (S.E.M.). Data were analysed by two-way ANOVA, and P-values reported for the effect of AICAR on the response to LPS.

Article Snippet: As AMPK inhibits mTOR, we also treated cells for 2 h with the mTOR complex 1 (mTORC1) inhibitor (5–500 nM rapamycin; Merck) or the mTORC1/2 inhibitor (10–150 nM Torin 1, 1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl) phenyl]-9 (3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one; Bio-Techne, Abingdon, UK), as described previously ( Oshiro et al. 2004 , Thoreen et al. 2009 ).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, MTT Assay

Journal: Reproduction (Cambridge, England)

Article Title: Manipulating bovine granulosa cell energy metabolism limits inflammation

doi: 10.1530/REP-20-0554

Figure Lengend Snippet: Intracellular metabolic sensors and granulosa cell inflammation. Granulosa cells from 4–8 mm (A, B and C) or > 8.5 mm diameter ovarian follicles (D, E and F) were cultured for 2 h with vehicle, 1 mM AICAR, 500 nM rapamycin, 50 nM Torin 1, or 1 μM dexamethasone; followed by a 24 h challenge with control medium (open circle) or medium containing 1 μg/mL LPS (closed circle), in the presence of the treatments. Supernatant IL-1α (A and D), IL-1β (B and E) and IL-8 (C and F) was measured by ELISA. Data from independent experiments are presented as dot plots, with bars indicating the mean (S.E.M.). Data were analysed by two-way ANOVA, with Dunnett’s post hoc test, and P-values differ between vehicle and treatments for the response to LPS.

Article Snippet: As AMPK inhibits mTOR, we also treated cells for 2 h with the mTOR complex 1 (mTORC1) inhibitor (5–500 nM rapamycin; Merck) or the mTORC1/2 inhibitor (10–150 nM Torin 1, 1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl) phenyl]-9 (3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one; Bio-Techne, Abingdon, UK), as described previously ( Oshiro et al. 2004 , Thoreen et al. 2009 ).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Reproduction (Cambridge, England)

Article Title: Manipulating bovine granulosa cell energy metabolism limits inflammation

doi: 10.1530/REP-20-0554

Figure Lengend Snippet: Rapamycin and Torin 1 reduce the phosphorylation of p70S6K. Granulosa cells from 4–8 mm (A) or > 8.5 mm diameter ovarian follicles (B) were treated for 24 h in culture medium with vehicle, 1 μg/mL LPS, 1 mM 2-deoxy-D-glucose (2-DG), 1 mM AICAR, 500 nM rapamycin or 50 nM Torin 1, and the phosphorylation of p70 S6 kinase (p70S6K) analysed by Western blot. Left panels show representative Western blots of phosphorylated p70S6K, total p70S6K and β-actin (Supplementary Fig. 1); right panels provide quantification of band densities for phosphorylated p70S6K relative to total p70S6K. Data from independent experiments are presented as dot plots, with bars indicating the mean (S.E.M.). Data were analysed by one-way ANOVA, with Dunnett’s post hoc test, and P values reported for differences between the vehicle and treatments.

Article Snippet: As AMPK inhibits mTOR, we also treated cells for 2 h with the mTOR complex 1 (mTORC1) inhibitor (5–500 nM rapamycin; Merck) or the mTORC1/2 inhibitor (10–150 nM Torin 1, 1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl) phenyl]-9 (3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one; Bio-Techne, Abingdon, UK), as described previously ( Oshiro et al. 2004 , Thoreen et al. 2009 ).

Techniques: Western Blot

Journal: Reproduction (Cambridge, England)

Article Title: Manipulating bovine granulosa cell energy metabolism limits inflammation

doi: 10.1530/REP-20-0554

Figure Lengend Snippet: Torin 1 limits LPS-stimulated inflammation in granulosa cells. Granulosa cells from 4–8 mm (A, B, C and D, I, J, K, L) or > 8.5 mm diameter ovarian follicles (E, F. G, H, M, N, O, P) were treated for 2 h with culture medium containing vehicle (V), or the indicated final concentrations of Torin 1 or rapamycin; followed by a 24 h challenge with control medium or 1 μg/mL LPS, in the presence of the treatments. Supernatant IL-1α, (A, E, I, M), IL-1β (B, F, J, N) and IL-8 (C, G, K, O) was measured by ELISA; cell viability was assessed by MTT assay (D, H, L, P). Data from independent experiments are presented as dot plots, with bars indicating the mean (S.E.M.). Data were analysed by one-way ANOVA, and P_values are reported for the effect of treatment on the LPS response.

Article Snippet: As AMPK inhibits mTOR, we also treated cells for 2 h with the mTOR complex 1 (mTORC1) inhibitor (5–500 nM rapamycin; Merck) or the mTORC1/2 inhibitor (10–150 nM Torin 1, 1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl) phenyl]-9 (3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one; Bio-Techne, Abingdon, UK), as described previously ( Oshiro et al. 2004 , Thoreen et al. 2009 ).

Techniques: Enzyme-linked Immunosorbent Assay, MTT Assay

Journal: Reproduction (Cambridge, England)

Article Title: Manipulating bovine granulosa cell energy metabolism limits inflammation

doi: 10.1530/REP-20-0554

Figure Lengend Snippet: Torin 1 did not alter LPS-stimulated phosphorylation of MAPK. Granulosa cells from 4–8 mm (A) or > 8.5 mm diameter ovarian follicles (B) were treated with vehicle or 50 nM Torin 1 for 2 h and then challenge with 1 μg/mL LPS for the indicated times, and the phosphorylation of ERK1/2 and JNK analysed by Western blot. Upper panels show representative Western blots of phosphorylated and total ERK1/2, phosphorylated and total JNK, and β-actin (Supplementary Fig. 1); lower panels provide quantification of band densities for phosphorylated relative to total ERK1/2 and JNK. Data are presented as mean (S.E.M.) from four independent experiments; statistical significance was determined using two-way ANOVA and P-values reported.

Article Snippet: As AMPK inhibits mTOR, we also treated cells for 2 h with the mTOR complex 1 (mTORC1) inhibitor (5–500 nM rapamycin; Merck) or the mTORC1/2 inhibitor (10–150 nM Torin 1, 1-[4-[4-(1-Oxopropyl)-1-piperazinyl]-3-(trifluoromethyl) phenyl]-9 (3-quinolinyl)-benzo[h]-1,6-naphthyridin-2(1H)-one; Bio-Techne, Abingdon, UK), as described previously ( Oshiro et al. 2004 , Thoreen et al. 2009 ).

Techniques: Western Blot

Journal: Cell Reports Medicine

Article Title: Overcoming Adaptive Resistance to KRAS and MEK Inhibitors by Co-targeting mTORC1/2 Complexes in Pancreatic Cancer

doi: 10.1016/j.xcrm.2020.100131

Figure Lengend Snippet:

Article Snippet: Torin-1 mTORC1/2 inhibitor , Selleck Chemicals , S2827; CAS: 1222998-36-8.

Techniques: Negative Control, Plasmid Preparation, Recombinant, Staining, Derivative Assay, Sequencing, shRNA, Control, Luciferase, Software, Imaging