Journal: bioRxiv

Article Title: mTORC2 coordinates the leading and trailing edge cytoskeletal programs during neutrophil migration

doi: 10.1101/2022.03.25.484773

Figure Lengend Snippet: (A) mTORC2 connects membrane stretch to regulation of front (magenta) and back (green) polarity programs, but how mTORC2 is activated (purely mechanical or requires biochemical co-inputs) and what aspect of mTORC2 activation (kinase-dependent vs independent roles) regulates these polarity signals is not understood. (B) To dissect the roles of kinase-dependent and kinase-independent roles of mTORC2, we generated individual CRISPR-Cas9 knockout lines of key components of the complex: Rictor (which scaffolds and aids structural integrity of the complex) or mSin1 (which primarily aids kinase activity). Additionally, mTOR Kinase inhibitors (here KU) would phenocopy mSin1 KO defects. (C) Representative immunoblots of wildtype (WT) HL-60 cells and gene-edited Rictor KO (top) and mSin1 KO (bottom) clonal HL-60 line to validate the loss of Rictor or mSin1 protein expression. GAPDH was used as a loading control. (D) Perturbation of mTORC2 activities in Rictor KO (n=3; red), mSin1KO(n=3; blue) and via mTOR Kinase inhibitor (KU; n=3; green) all led to defective transwell migration towards chemoattractant 20nM fMLP in comparison to WT cells (n=6; black). Mean ± SEM is plotted, n indicates independent replicates. (E) Schematic shows neutrophil-like dHL60 cell moving under an agarose (2%) overlay with uniform chemoattractant (25 nM fMLP). Randomly-chosen representative tracks (15 each) of wildtype (WT), Rictor KO, or mSin1KO cells over a 12 min observation window; axes show x-y displacement in μm. Rictor KO cells migrate poorly and have markedly shorter displacements. (F, G) Box plots (with kernel smooth distribution curve) show mean speed (F) and persistence (G; ratio of displacement/distance) averaged over individual tracks. Both Rictor KO and mSin1 KO cells shows a significant reduction (p < 0.01; one-way ANOVA with Tukey-means comparison) in migration speed compared to Wildtype. However, only Rictor KO show a significant decrease in the persistence (p < 0.01; one-way ANOVA with Tukey-means comparison). N = 294 (WT), 138 (RictorKO) and 165 (mSin1KO) tracks from individual cells pooled across 3 independent experiments. For box plots, median is indicated by the line, inter-quartile range (IQR) sets the box width and error bars indicate 10-90 th percentile. (H) Schematic highlights the phenotypes observed for mSin1KO and Rictor KO cells. Kinase-dependent roles of mTORC2 appear to regulate speed whereas kinase-independent role regulates both persistence and speed.

Article Snippet: The following primary antibodies were used for the study; phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197) (Cell Signaling #2605), PAK2 (3B5) (Cell Signaling #4825), phospho-Akt (Ser473; D9E) XP (Cell Signaling #4060), Akt (pan; 40D4; Cell Signaling #2920S), Rictor (Bethyl #A300-458A), mSin1 (Bethyl # A300-910A), and GAPDH Loading Control Antibody GA1R (ThermoFisher).

Techniques: Activation Assay, Generated, CRISPR, Knock-Out, Activity Assay, Western Blot, Expressing, Migration

Journal: bioRxiv

Article Title: mTORC2 coordinates the leading and trailing edge cytoskeletal programs during neutrophil migration

doi: 10.1101/2022.03.25.484773

Figure Lengend Snippet: (A,B) Sequence validation to infer CRISPR indel edits in the clonal Rictor KO (A) and mSin1 KO (B) lines assayed here. The green bar above both sequences shows the gRNA target sequence. Both lines have deletions that lead to a frame shift, nonsense, and termination following Exon 2. (C, D) mTORC2 kinase activity assayed by immunoblots of phospho Akt and total Akt levels before (basal,-) and 3 min after chemoattractant (fMLP, +) addition. Representative western blots and quantification (D) shows significant loss of mTORC2 kinase activity for Rictor KO, mSin1KO and mTOR drug KU (assayed by pAkt immunoblots). Plots (D) show pAkt/totalAkt ratio (mean ± SEM from three independent trials) normalised with values obtained for the wildtype for each trial. (E) Schematic shows a neutrophil-like dHL60 cell undergoing unconfined motility on glass coated with fibronectin in presence of uniform chemoattractant fMLP. Randomly chosen representative tracks (15 each) of wildtype (WT), Rictor KO, or mSin1KO cells over a 12 min observation window; axes show x-y displacement in μm. (F, G) Box plots (with kernel smooth distribution curve) show mean speed (D) and persistence (E; ratio of displacement/distance) averaged over individual tracks. Both Rictor KO and mSin1 KO cells shows normal persistence and speed in unconfined 2D migration (p < 0.01; one-way ANOVA with Tukey-means comparison). N = 203 (WT), 338 (RictorKO) and 392 (mSin1KO) tracks from individual cells pooled across 2 independent experiments. For box plots, median is indicated by the line, inter-quartile range sets the box width and error bars indicate 10-90 th percentile.

Article Snippet: The following primary antibodies were used for the study; phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197) (Cell Signaling #2605), PAK2 (3B5) (Cell Signaling #4825), phospho-Akt (Ser473; D9E) XP (Cell Signaling #4060), Akt (pan; 40D4; Cell Signaling #2920S), Rictor (Bethyl #A300-458A), mSin1 (Bethyl # A300-910A), and GAPDH Loading Control Antibody GA1R (ThermoFisher).

Techniques: Sequencing, CRISPR, Activity Assay, Western Blot, Migration

Journal: The Journal of Cell Biology

Article Title: Inhibition of class I PI3K enhances chaperone-mediated autophagy

doi: 10.1083/jcb.202001031

Figure Lengend Snippet: Inhibiting p110 activates CMA in NIH3T3 cells. (A) Western blots showing effects of selected doses of buparlisib (2 µM), pictilisib (500 nM), and copanlisib (50 nM) on phosphorylation of Akt, MAPKAP1, and GFAP. Blots are representative of six experimental replicates. DMSO is the solvent control for buparlisib and pictilisib. TFA is the solvent control for copanlisib. (B) Quantification of GFAP phosphorylation change in response to p110 inhibitors, as shown in A, n = 6. For C–H, data points are pooled from at least three independent experiments. (C and D) 10 h buparlisib treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 92 cells; buparlisib = 83 cells. (E and F) 10 h pictilisib treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 71 cells; pictilisib = 86 cells. (G and H) 10 h copanlisib treatment induces the accumulation of Dendra2 CMA reporter puncta. TFA = 132 cells; copanlisib = 116 cells. (I–K) Effects of buparlisib, pictilisib, and copanlisib on macroautophagy, as measured by LC3-II flux, n = 6. For all experiments, cells were maintained in complete growth medium with 10% serum. P values written above brackets are derived from unpaired t tests. **, P < 0.01 by unpaired t test. For charts in I – K, P INT is the interaction term P value from a two-way ANOVA. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin; Bup, buparlisib; Cop, copanlisib; Pic, pictilisib; TFA, trifluoroacetic acid.

Article Snippet: Antibodies were acquired as follows: ACACA/ACC1 (Cell Signaling Technology; 4190S; rabbit host), ACTB/β-Actin (Cell Signaling Technology; 8457L; rabbit host), ALDOB (AbCam; 137628; rabbit host), AKT (Cell Signaling Technology; 4691S; rabbit host), pT308 AKT (Cell Signaling Technology; 13038S; rabbit host), pS473 AKT (Cell Signaling Technology; 4060S; rabbit host), ATG5 (Cell Signaling Technology; 12994S; rabbit host), CIP2A (Santa Cruz; sc-80659; mouse host), CTSD/cathepsin D (AbCam; 75852; rabbit host), EIF2A (Cell Signaling Technology; 9722S; rabbit host), EIF5 (Cell Signaling Technology; 13894S; rabbit host), ENO1 (Cell Signaling Technology; 3810S; rabbit host), GAPDH (Cell Signaling Technology; 2118S; rabbit host), GSK3β (Cell Signaling Technology; 12456S; rabbit host), pS9 GSK3β (Cell Signaling Technology; 9323S; rabbit host), GFAP (AbCam; 7260; rabbit host), pS8 GFAP (Thermo Fisher Scientific; PA5-12991; rabbit host), HSPA8/Hsc70 (AbCam; 154415; rabbit host), IDH1 (AbCam; 172964; rabbit host), IRS1 (Cell Signaling Technology; 2382S; rabbit host) LAMP1 (AbCam; 24170; rabbit host), LAMP2A (AbCam; 125068; rabbit host), MAP1LC3B/LC3 (Cell Signaling Technology; 2775S; rabbit host), MAPKAP1/mSIN1 (AbCam; 64188; rabbit host), pT86 MAPKAP1/mSIN1 (Cell Signaling Technology; 14716S; rabbit host), MAPT/Tau (Cell Signaling Technology; 46687S; rabbit host), OXPHOS (oxidative phosphorylation complex) cocktail (AbCam; 110413; mouse host), PPID/cyclophilin 40 (AbCam; 181983; rabbit host), and SQSTM1/p62 (Cell Signaling Technology; 5114S; rabbit host).

Techniques: Western Blot, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Inhibition of class I PI3K enhances chaperone-mediated autophagy

doi: 10.1083/jcb.202001031

Figure Lengend Snippet: Inhibiting PDPK1 activates CMA in NIH3T3 cells. (A) Western blots showing effects of selected doses of BX795 (5 µM), BX912 (15 µM), and GSK2334470 (5 µM) on phosphorylation of Akt, MAPKAP1, and GFAP. Blots are representative of six experimental replicates. DMSO is the solvent control for all of the drugs. (B) Quantification of GFAP phosphorylation change in response to PDPK1 inhibitors, as shown in A, n = 6. In A and B, GSK2334470 is abbreviated for space, as GSK233. For C – H, data points are pooled from at least three independent experiments. (C and D) 10 h BX795 treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 88 cells; BX795 = 90 cells. (E and F) 10 h BX912 treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 155 cells; BX912 = 120 cells. (G and H) 10 h GSK2334470 treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 145 cells; GSK2334470 = 144 cells. (I–K) Effects of BX795, BX912, and GSK233470 on macroautophagy, as measured by LC3-II flux, n = 6. For all experiments, cells were maintained in complete growth medium with 10% serum. P values written above brackets are derived from unpaired t tests. **, P < 0.01 by unpaired t test. For charts in I–K, P INT is the interaction term P value from a two-way ANOVA. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin.

Article Snippet: Antibodies were acquired as follows: ACACA/ACC1 (Cell Signaling Technology; 4190S; rabbit host), ACTB/β-Actin (Cell Signaling Technology; 8457L; rabbit host), ALDOB (AbCam; 137628; rabbit host), AKT (Cell Signaling Technology; 4691S; rabbit host), pT308 AKT (Cell Signaling Technology; 13038S; rabbit host), pS473 AKT (Cell Signaling Technology; 4060S; rabbit host), ATG5 (Cell Signaling Technology; 12994S; rabbit host), CIP2A (Santa Cruz; sc-80659; mouse host), CTSD/cathepsin D (AbCam; 75852; rabbit host), EIF2A (Cell Signaling Technology; 9722S; rabbit host), EIF5 (Cell Signaling Technology; 13894S; rabbit host), ENO1 (Cell Signaling Technology; 3810S; rabbit host), GAPDH (Cell Signaling Technology; 2118S; rabbit host), GSK3β (Cell Signaling Technology; 12456S; rabbit host), pS9 GSK3β (Cell Signaling Technology; 9323S; rabbit host), GFAP (AbCam; 7260; rabbit host), pS8 GFAP (Thermo Fisher Scientific; PA5-12991; rabbit host), HSPA8/Hsc70 (AbCam; 154415; rabbit host), IDH1 (AbCam; 172964; rabbit host), IRS1 (Cell Signaling Technology; 2382S; rabbit host) LAMP1 (AbCam; 24170; rabbit host), LAMP2A (AbCam; 125068; rabbit host), MAP1LC3B/LC3 (Cell Signaling Technology; 2775S; rabbit host), MAPKAP1/mSIN1 (AbCam; 64188; rabbit host), pT86 MAPKAP1/mSIN1 (Cell Signaling Technology; 14716S; rabbit host), MAPT/Tau (Cell Signaling Technology; 46687S; rabbit host), OXPHOS (oxidative phosphorylation complex) cocktail (AbCam; 110413; mouse host), PPID/cyclophilin 40 (AbCam; 181983; rabbit host), and SQSTM1/p62 (Cell Signaling Technology; 5114S; rabbit host).

Techniques: Western Blot, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Inhibition of class I PI3K enhances chaperone-mediated autophagy

doi: 10.1083/jcb.202001031

Figure Lengend Snippet: Effects of PI3K inhibitors on CMA and macroautophagy in AML12 cells. (A–C) Dose curves in AML12 cells for buparlisib, pictilisib, and autophinib, respectively. (D) Western blots demonstrating the effects of buparlisib (5 µM), pictilisib (2 µM), and autophinib (5 µM) on phosphorylation of Akt, MAPKAP1, and GFAP. Blots are representative of six experimental replicates. DMSO is the solvent control for all drugs. (E and F) 10 h treatment with buparlisib or pictilisib induces the accumulation of Dendra2 CMA reporter puncta, but treatment with autophinib does not. Data were pooled from at least three independent experiments. DMSO = 202 cells, buparlisib = 122 cells, pictilisib = 104 cells, and autophinib = 95 cells. (G–I) Effects of buparlisib, pictilisib, and autophinib on macroautophagy, as measured by LC3-II flux, n = 6. For all experiments, cells were maintained in complete growth medium (see Materials and methods). P values written above brackets are derived from unpaired t tests. Two-way ANOVA tables are displayed below graphs where appropriate. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin; Aph, autophinib; Bup, buparlisib; Pic, pictilisib.

Article Snippet: Antibodies were acquired as follows: ACACA/ACC1 (Cell Signaling Technology; 4190S; rabbit host), ACTB/β-Actin (Cell Signaling Technology; 8457L; rabbit host), ALDOB (AbCam; 137628; rabbit host), AKT (Cell Signaling Technology; 4691S; rabbit host), pT308 AKT (Cell Signaling Technology; 13038S; rabbit host), pS473 AKT (Cell Signaling Technology; 4060S; rabbit host), ATG5 (Cell Signaling Technology; 12994S; rabbit host), CIP2A (Santa Cruz; sc-80659; mouse host), CTSD/cathepsin D (AbCam; 75852; rabbit host), EIF2A (Cell Signaling Technology; 9722S; rabbit host), EIF5 (Cell Signaling Technology; 13894S; rabbit host), ENO1 (Cell Signaling Technology; 3810S; rabbit host), GAPDH (Cell Signaling Technology; 2118S; rabbit host), GSK3β (Cell Signaling Technology; 12456S; rabbit host), pS9 GSK3β (Cell Signaling Technology; 9323S; rabbit host), GFAP (AbCam; 7260; rabbit host), pS8 GFAP (Thermo Fisher Scientific; PA5-12991; rabbit host), HSPA8/Hsc70 (AbCam; 154415; rabbit host), IDH1 (AbCam; 172964; rabbit host), IRS1 (Cell Signaling Technology; 2382S; rabbit host) LAMP1 (AbCam; 24170; rabbit host), LAMP2A (AbCam; 125068; rabbit host), MAP1LC3B/LC3 (Cell Signaling Technology; 2775S; rabbit host), MAPKAP1/mSIN1 (AbCam; 64188; rabbit host), pT86 MAPKAP1/mSIN1 (Cell Signaling Technology; 14716S; rabbit host), MAPT/Tau (Cell Signaling Technology; 46687S; rabbit host), OXPHOS (oxidative phosphorylation complex) cocktail (AbCam; 110413; mouse host), PPID/cyclophilin 40 (AbCam; 181983; rabbit host), and SQSTM1/p62 (Cell Signaling Technology; 5114S; rabbit host).

Techniques: Western Blot, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Inhibition of class I PI3K enhances chaperone-mediated autophagy

doi: 10.1083/jcb.202001031

Figure Lengend Snippet: Effects of PI3K inhibitors on CMA and macroautophagy in mIMCD3 cells. (A–C) Dose curves in mIMCD3 cells for buparlisib, pictilisib, and autophinib, respectively. (D) Western blots demonstrating the effects of buparlisib (1 µM), pictilisib (1 µM), and autophinib (5 µM) on phosphorylation of Akt, MAPKAP1, and GFAP. Blots are representative of six experimental replicates. DMSO is the solvent control for all drugs. (E and F) 10 h treatment with buparlisib or pictilisib induces the accumulation of Dendra2 CMA reporter puncta, but treatment with autophinib does not. Data were pooled from at least three independent experiments. DMSO = 373 cells, buparlisib = 85 cells, pictilisib = 112 cells, and autophinib = 204 cells. (G–I) Effects of buparlisib, pictilisib, and autophinib on macroautophagy, as measured by LC3-II flux, n = 6. For all experiments, cells were maintained in complete growth medium (see Materials and methods). P values written above brackets are derived from unpaired t tests. Two-way ANOVA tables are displayed below graphs where appropriate. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin; Aph, autophinib; Bup, buparlisib; Pic, pictilisib.

Article Snippet: Antibodies were acquired as follows: ACACA/ACC1 (Cell Signaling Technology; 4190S; rabbit host), ACTB/β-Actin (Cell Signaling Technology; 8457L; rabbit host), ALDOB (AbCam; 137628; rabbit host), AKT (Cell Signaling Technology; 4691S; rabbit host), pT308 AKT (Cell Signaling Technology; 13038S; rabbit host), pS473 AKT (Cell Signaling Technology; 4060S; rabbit host), ATG5 (Cell Signaling Technology; 12994S; rabbit host), CIP2A (Santa Cruz; sc-80659; mouse host), CTSD/cathepsin D (AbCam; 75852; rabbit host), EIF2A (Cell Signaling Technology; 9722S; rabbit host), EIF5 (Cell Signaling Technology; 13894S; rabbit host), ENO1 (Cell Signaling Technology; 3810S; rabbit host), GAPDH (Cell Signaling Technology; 2118S; rabbit host), GSK3β (Cell Signaling Technology; 12456S; rabbit host), pS9 GSK3β (Cell Signaling Technology; 9323S; rabbit host), GFAP (AbCam; 7260; rabbit host), pS8 GFAP (Thermo Fisher Scientific; PA5-12991; rabbit host), HSPA8/Hsc70 (AbCam; 154415; rabbit host), IDH1 (AbCam; 172964; rabbit host), IRS1 (Cell Signaling Technology; 2382S; rabbit host) LAMP1 (AbCam; 24170; rabbit host), LAMP2A (AbCam; 125068; rabbit host), MAP1LC3B/LC3 (Cell Signaling Technology; 2775S; rabbit host), MAPKAP1/mSIN1 (AbCam; 64188; rabbit host), pT86 MAPKAP1/mSIN1 (Cell Signaling Technology; 14716S; rabbit host), MAPT/Tau (Cell Signaling Technology; 46687S; rabbit host), OXPHOS (oxidative phosphorylation complex) cocktail (AbCam; 110413; mouse host), PPID/cyclophilin 40 (AbCam; 181983; rabbit host), and SQSTM1/p62 (Cell Signaling Technology; 5114S; rabbit host).

Techniques: Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: c-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

doi: 10.1074/jbc.RA118.003926

Figure Lengend Snippet: LPA induces mTORC2 signaling-associated mSin1 in mesenchymal cells. A, nonfibrotic MCs were pretreated with the mTORC inhibitor AZD8055 (250 nm for 30 min) and then exposed to LPA (10 μm for 6 h). Protein expression of the total and the phosphorylated forms of AKT at Thr-308 and Ser-473 were measured by immunoblotting. B, cell lysates isolated from normal nonfibrotic MCs were immunoblotted for antibodies against mTORC2-associated mSin1 and rictor, mTORC1-associated raptor, and GAPDH (loading control (C)). C, quantitative densitometry of the mTORC2 binding partner mSin1 was analyzed at 6 and 16 h in response to LPA. Values are means ± S.D., n = 4–6. *, p < 0.05; **, p < 0.01. D, nonfibrotic MCs were exposed to TGF-β (2 ng/ml) or IL-13 (10 ng/ml) for 16 h. Lysates were immunoblotted against mSin1. Quantitative densitometry was normalized using GAPDH (loading control). Values are means ± S.D., n = 9. *, p < 0.05; **, p < 0.01. Statistics: one-way ANOVA; post hoc: Sidak (C and D).

Article Snippet: The other antibodies utilized were as follows: phosphorylated p70-S6kinase (Thr-389, 9234), p70-S6Kinase (2708), phosphorylated 4E-BP1 (Thr-37/46, 2855), total 4E-BP1 (9644), phosphorylated AKT (Ser-473, 4058), phosphorylated AKT (Thr-308, 9275), total AKT (9272), phosphorylated TSC2 (Thr-1462, 3611), total TSC2 (3635), total FOXO3a (12829), ubiquitin (3936), rictor (9476), raptor (2280), phosphorylated mTOR (Ser-2481, 2974), total mTOR (2972), JNK2 (9258), and mSin1 (12860) (all from Cell Signaling Technology, Boston, MA) and phosphorylated FOXO3a (Ser-253, ab154786, Abcam).

Techniques: Expressing, Western Blot, Isolation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: c-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

doi: 10.1074/jbc.RA118.003926

Figure Lengend Snippet: mSin1 is required for mTORC regulation and fibrotic functions of mesenchymal cells. A, nonfibrotic MCs (n = 4) were transfected with scrambled or mSIN1-specific siRNA and then treated with LPA (10 μm for 6 h). Densitometry of the replicates was analyzed. Values are means ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B–D, cell lysates were subjected to immunoblot analysis for phosphorylated and total forms of AKT (Ser-473, B), p70S6K1 (Thr-389, C), and 4E-BP1 (Thr-37/46, D). Densitometry of the replicates was analyzed. Values are means ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, nonfibrotic MCs and fibrotic MCs (n = 10) were analyzed for mSin1 protein expression by immunoblotting, and the densitometric analysis is presented as ***, p < 0.001. F, mesenchymal cells derived from fibrotic lung allografts (fibrotic MCs, n = 6) were transfected with scrambled or mSIN1-specific siRNA, and the lysates were immunoblotted with antibodies against mSin1, collagen I, phosphorylated and total forms of AKT (Ser-473), TSC2 (Thr-1462), FOXO3a (Ser-253), p70S6K1 (Thr-389), and 4E-BP1 (Thr-37/46) using GAPDH as a loading control. G, fibrotic MCs (n = 10) were transfected with siRNA specific to LPAR1 or the scrambled control, and the lysates were immunoblotted with antibodies against LPA1, collagen I, and mSin1 as well as the phosphorylated and total forms of AKT (Ser-473), p70S6K1 (Thr-389), and 4E-BP1 (Thr-37/46) using GAPDH as a loading control. Representative blots are shown.

Article Snippet: The other antibodies utilized were as follows: phosphorylated p70-S6kinase (Thr-389, 9234), p70-S6Kinase (2708), phosphorylated 4E-BP1 (Thr-37/46, 2855), total 4E-BP1 (9644), phosphorylated AKT (Ser-473, 4058), phosphorylated AKT (Thr-308, 9275), total AKT (9272), phosphorylated TSC2 (Thr-1462, 3611), total TSC2 (3635), total FOXO3a (12829), ubiquitin (3936), rictor (9476), raptor (2280), phosphorylated mTOR (Ser-2481, 2974), total mTOR (2972), JNK2 (9258), and mSin1 (12860) (all from Cell Signaling Technology, Boston, MA) and phosphorylated FOXO3a (Ser-253, ab154786, Abcam).

Techniques: Transfection, Western Blot, Expressing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: c-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

doi: 10.1074/jbc.RA118.003926

Figure Lengend Snippet: LPA-mediated induction of the mTORC2 binding partner mSin1 depends on JNK. A, nonfibrotic MCs (n = 8) were pretreated with inhibitors specific to LPA1 (VPC12249), Gi (PTX), or PI3K (LY294002) and then stimulated with LPA (10 μm for 6 h). Cell lysates were immunoblotted with antibodies against phosphorylated and total forms of AKT, mSin1, and GAPDH (loading control). Densitometry analysis of mSin1 was expressed as means ± S.D. **, p < 0.01; n = 9. B, nonfibrotic MCs (n = 9) were pretreated with inhibitors specific to p38MAPK (SB203580), ERK (U0126), and JNK (SP600125) and then stimulated with LPA (10 μm for 6 h). Cell lysates were immunoblotted with antibodies against the phosphorylated and total forms of AKT, mSin1, and GAPDH (loading control). Densitometry analysis of mSin1 was expressed as means ± S.D. **, p < 0.01; *, p < 0.05. C, nonfibrotic MCs (n = 9) were pretreated with a JNK inhibitor (SP600125), followed by stimulation with IL-13 (10 ng/ml) or TGF-β (2 ng/ml) for 16 h. Cell lysates were analyzed for mSin1 by immunoblotting. Densitometry analysis of mSin1 was expressed as means ± S.D. ***, p < 0.001. Statistics: one-way ANOVA; post hoc: Sidak. D, nonfibrotic MCs were transfected with scrambled or JNK1-specific siRNA and then stimulated with LPA (10 μm for 6 h). Cell lysates were immunoblotted with antibodies against mSin1, collagen I, JNK1/2, or phosphorylated and total forms of AKT (Ser-473), p70S6K1 (Thr-389), and 4E-BP1 (Thr-37/46) using GAPDH as a loading control. Representative blots are shown.

Article Snippet: The other antibodies utilized were as follows: phosphorylated p70-S6kinase (Thr-389, 9234), p70-S6Kinase (2708), phosphorylated 4E-BP1 (Thr-37/46, 2855), total 4E-BP1 (9644), phosphorylated AKT (Ser-473, 4058), phosphorylated AKT (Thr-308, 9275), total AKT (9272), phosphorylated TSC2 (Thr-1462, 3611), total TSC2 (3635), total FOXO3a (12829), ubiquitin (3936), rictor (9476), raptor (2280), phosphorylated mTOR (Ser-2481, 2974), total mTOR (2972), JNK2 (9258), and mSin1 (12860) (all from Cell Signaling Technology, Boston, MA) and phosphorylated FOXO3a (Ser-253, ab154786, Abcam).

Techniques: Binding Assay, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: c-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

doi: 10.1074/jbc.RA118.003926

Figure Lengend Snippet: JNK1 activation induces mSin1 expression and mTORC2 stabilization. A, nonfibrotic MCs (n = 6) were infected with a lentivirus containing an empty vector or a vector that expresses constitutively active JNK1 tagged with FLAG and then treated with the mTOR inhibitor AZD8055 (250 nm for 24 h). Cell lysates were immunoblotted against mSin1, collagen I, GAPDH (loading control), FLAG, and the phosphorylated and total forms of Jun (Ser-73), AKT (Ser-473), p70S6K1(Thr-389), 4E-BP1 (Thr-37/46), and mTOR (Ser-2481). B, nonfibrotic MCs (n = 6) were infected with a lentivirus containing an empty vector or vector that expresses constitutively active JNK1, followed by transfection with scrambled or mSIN1-specific siRNA. Cell lysates were immunoblotted with antibodies as described in A. C, nonfibrotic MCs (n = 8) were pretreated with 10 μm JNK inhibitor (SP600125) and then stimulated with LPA (10 μm for 6 h). Cell lysates were immunoblotted with antibodies against mSin1, collagen I, and phosphorylated and total forms of JNK1/2 (Thr-183/Tyr-185), AKT (Ser-473), and mTOR (Ser-2481) as well as rictor, raptor, and GAPDH. Cell lysates were immunoprecipitated using antibodies against mSin1, rictor, or mTOR. Each of the immunoprecipitates was analyzed for rictor, mSin1, phosphorylated mTOR (Ser-2481), and total mTOR. Representative blots are shown for each experiment.

Article Snippet: The other antibodies utilized were as follows: phosphorylated p70-S6kinase (Thr-389, 9234), p70-S6Kinase (2708), phosphorylated 4E-BP1 (Thr-37/46, 2855), total 4E-BP1 (9644), phosphorylated AKT (Ser-473, 4058), phosphorylated AKT (Thr-308, 9275), total AKT (9272), phosphorylated TSC2 (Thr-1462, 3611), total TSC2 (3635), total FOXO3a (12829), ubiquitin (3936), rictor (9476), raptor (2280), phosphorylated mTOR (Ser-2481, 2974), total mTOR (2972), JNK2 (9258), and mSin1 (12860) (all from Cell Signaling Technology, Boston, MA) and phosphorylated FOXO3a (Ser-253, ab154786, Abcam).

Techniques: Activation Assay, Expressing, Infection, Plasmid Preparation, Transfection, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: c-Jun N-terminal kinase (JNK)–mediated induction of mSin1 expression and mTORC2 activation in mesenchymal cells during fibrosis

doi: 10.1074/jbc.RA118.003926

Figure Lengend Snippet: JNK mediates posttranslational regulation of mSin1 induction. A, nonfibrotic MCs (n = 6) were stimulated with LPA (10 μm for 6 h). RNA lysates were analyzed for mSIN1 by qPCR, using β-actin as the endogenous control. B, nonfibrotic MCs (n = 9) were pretreated with actinomycin D (ActD, 5 μg/ml for 30 min), followed by stimulation with LPA. Cell lysates were immunoblotted for antibodies against mSin1 and GAPDH (loading control). **, p < 0.01. Statistics: one-way ANOVA; post hoc: Sidak. C, nonfibrotic MCs (n = 5) were pretreated with CHX (10 μm) for the indicated periods of time. Protein lysates were subjected to a chase assay and analyzed for mSin1. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, nonfibrotic MCs (n = 12) were treated with the proteasomal inhibitor MG132 (10 μm for 16 h). Protein lysates were immunoblotted with antibodies against mSin1 and GAPDH (loading control). Densitometric values are represented as means ± S.D. ***, p < 0.001. E, nonfibrotic MCs (n = 14) were pretreated with a proteasomal inhibitor (MG132) or JNK inhibitor (SP600125) or both, followed by stimulation with LPA. Cell lysates were analyzed for mSin1 and GAPDH (loading control). Densitometric values are represented as means ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistics: one-way ANOVA; post hoc: Sidak. F, nonfibrotic MCs (n = 3) were treated with the proteasomal inhibitor MG132 in the presence or absence of JNK inhibitor II (SP600125) with or without LPA stimulation for 16 h. Cell lysates were immunoprecipitated using antibodies against mSin1. mSin1 immunoprecipitates were immunoblotted with antibodies against pan-ubiquitin, mSin1, rictor, and total mTOR. Representative blots are shown.

Article Snippet: The other antibodies utilized were as follows: phosphorylated p70-S6kinase (Thr-389, 9234), p70-S6Kinase (2708), phosphorylated 4E-BP1 (Thr-37/46, 2855), total 4E-BP1 (9644), phosphorylated AKT (Ser-473, 4058), phosphorylated AKT (Thr-308, 9275), total AKT (9272), phosphorylated TSC2 (Thr-1462, 3611), total TSC2 (3635), total FOXO3a (12829), ubiquitin (3936), rictor (9476), raptor (2280), phosphorylated mTOR (Ser-2481, 2974), total mTOR (2972), JNK2 (9258), and mSin1 (12860) (all from Cell Signaling Technology, Boston, MA) and phosphorylated FOXO3a (Ser-253, ab154786, Abcam).

Techniques: Immunoprecipitation