Journal: PLoS ONE

Article Title: TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

doi: 10.1371/journal.pone.0042316

Figure Lengend Snippet: Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.

Article Snippet: Phospho-Akt (Ser473), phospho-Akt (thr-308), Akt, phospho-S6 kinase, S6 kinase, phospho-4EBP-1 (Thr-37/46) phospho-4EBP-1 (Ser-65) 4EBP-1, phospho-GSK3β, GSK3β, phospho-tuberin (Thr 1462), tuberin, phospho-PRAS40 (Thr 246), PRAS40, phospho-mTOR (Ser-2448) and mTOR antibodies were purchased from Cell Signaling, Boston, MA. siRNA pool of three oligonucleotides against PTEN mRNA, collagen II (α2) and PTEN antibodies were obtained from Santacruz, Delaware, CA.

Techniques: Transfection, Plasmid Preparation, Incubation

Journal: Journal of Cancer

Article Title: Torin2 inhibits the EGFR-TKI resistant Non-Small Lung Cancer cell proliferation through negative feedback regulation of Akt/mTOR signaling

doi: 10.7150/jca.37417

Figure Lengend Snippet: Torin2 inhibited the growth of EGFR-TKI resistant cell by down-regulated negative-feedback loop of Akt/mTOR signaling pathway. (A) Western blot analysis showed that level of phosphorylated S6 was reduced in PC9 cell treated with Erlotinib or Torin2, respectively. The level of phosphorylated Akt(T308) was activated by erlotinib, whereas Torin2 significantly diminished the phosphorylated Akt(T308) and Akt(S473) in a dose dependent manner. (B) In the PC9/E cell line, the expression of phosphorylated S6 and 4EBP-1 were obviously decreased in Torin2 treated group compared with erlotinib treatment. Torin2 also markedly down-regulated both phosphorylated Akt(S473) and Akt(T308) in PC9/E compared to erlotinib group.

Article Snippet: The following antibodies were used: BAX (#5023), BCL-2, (#4223), cleaved PARP (#5625), anti-Akt (#2920) and phospho-Akt at Ser473 (#9271), 4EBP-1 (#9459) and phospho-4EBP-1 (#2855), β-actin (#3700), anti-S6 (#2317) and phospho-S6 at Ser240/244 (#5364) (diluted with 5% BSA to 1: 1000) were obtained from Cell Signaling Technology, Beverly, MA, USA). p62/SQSTM1 (#H00008878-M01) from Abnova (Taipei City, Taiwan), and LC3 (L8918) and β-actin (#A5316) from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Impact of Ninjin’yoeito on frailty and short life in klotho-hypomorphic (kl/kl) mice

doi: 10.3389/fphar.2022.973897

Figure Lengend Snippet: The activities of myoprotein synthesis factors and myoproteolytic factors in 59 days old were evaluated by western-blotting analysis using the dissected gastrocnemius muscle. The detected bands are shown below the graph of quantified arbitrary unit. Factors related to muscle protein synthesis: phosphorylation of AKT1 at Ser473 (D) , phosphorylation of 4E-BP1 at Ser65/Thr37-46 (A) , phosphorylation of p70S6K at Thr389 (H) , the amount of PGC1α (F) ; degradation-related factors: the amount of Atrogin-1 (B) , the amount of Murf1 (G) , phosphorylation of FoxO1 Ser256 (C) , the amount of LC3II (E) . Data are indicated as mean and SEM values. Significant differences are presented on histograms: [* p < 0.05, ** p < 0.01, Dunnett’s contrast, WT ( n = 5), NT (8), 3% NYT (6), 5% NYT (4)].

Article Snippet: After blocking with 5% skim milk for 1 h, membranes were incubated with the primary antibodies anti-4E-BP1(pSer65) Ab (#9451 Cell Signaling Technology (CST), MA, United States), anti-Phospho-4EBP-1(Thr37/46)Ab (#9459; CST), anti-atrogin-l Ab (#9452; CST), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (#G8795; Sigma-Aldrich, MO, United States), anti-forkhead box O (FoxO)-1(pSer256) Ab (#g234; CST), anti-FoxO-1 Ab (#2880; CST), anti-Akt (pSer473) Ab (#4051; CST), anti-Akt1 Ab (#9272; CST), anti-muscle RING-finger protein (MURF)-1 Ab (#MP3401; ECM bioscience, KY, United States), anti-peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1) Ab (ab54481; Abcam, Cambridge, United Kingdom), anti-p70S6K Ab (#9234; CST), anti-p70S6K Ab (#2708; CST), anti-light chain 3B (LC3B) Ab (#L7543; Sigma) in 5% BSA/TBS for 8 h at 4°C.

Techniques: Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: MDSCs participate in the pathogenesis of diffuse pulmonary hemorrhage in murine lupus through mTOR-FoxO1 signaling

doi: 10.1016/j.bbrep.2022.101351

Figure Lengend Snippet: Activation of mTOR signaling pathway in MDSCs is involved in the occurrence of DPH (A) C57BL/6 mice (6–8 weeks old)(n = 11/group) received a single intraperitoneal injection of 0.5 ml pristane for 14 days to establish the DPH mouse model. Expression of p-4EBP, 4EBP1, p-S6, and S6 in MDSCs isolated from control and DPH mice spleen was detected by Western blot. (B) The density quantification of band intensities. (C) C57BL/6 mice (6–8 weeks old) (n = 11/group) received a single intraperitoneal injection of 0.5 ml pristane for 14 days, and metformin, INK128, and Rapamycin during induction. The lung tissues were observed for evaluating the symptoms of diffuse pulmonary hemorrhage, and statistical analysis of the proportion of three types of pulmonary hemorrhage symptoms in each group. (D) Morphological analysis of spleens of mice in different groups.

Article Snippet: Antibodies for p-S6 (Cat. No. 4858S), S6 (Cat. No. 2217S), p-4EBP-1 (Cat. No. 2855S), and 4EBP-1 (Cat. No. 9644T), and horseradish peroxides (HRP)-conjugated anti-rabbit IgG (Cat. No. 7074P2) for Western blot were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Injection, Expressing, Isolation, Western Blot

Journal: Communications Biology

Article Title: mTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling

doi: 10.1038/s42003-022-03653-w

Figure Lengend Snippet: a HAEC were treated with rapamycin (rapa) or torin 1 (torin) at indicated dose for 1 h prior to western blotting. *indicated the phosphorylated 4EBP-1. b , c NO levels of rapa ( b ) or torin-treated ( c ) HAEC were measured with colorimetric reaction assay. d , e At 48 h post transfection with control siRNA (sictrl), siRNA targeting RPTOR (siRPT), RICTOR (siRIC) or MTOR (siMTOR) , HAEC were submitted to western blot analysis ( d ) or NO measurement with colorimetric reaction assay ( e ) ( n = 5–7). Shown images are representative blots of at least five experiments. * p < 0.05; ** p < 0.01 vs. ctrl or sictrl; unpaired two-tailed t test ( b , c ) or one-way ANOVA with Dunnett’s post-test ( e ).

Article Snippet: Antibodies targeting mTOR (#2983), Rictor (#2114), Raptor (#2280), p-eNOS (S1177) (#9570), IRF-1 (#8478), AP2α (#3215), p-p70S6K (Ser389) (#9234), p70S6K (#9202), p-4EBP-1 (T37/46) (#2855), 4EBP-1 (#9452), p-AKT (S473) (#4060), AKT (#4691), p-p38 (Thr180/Tyr182) (#4511), p38 (#8690), p-ERK (Thr202/Tyr204) (#4370), ERK (#4695), p-SAPK/JNK (Thr183/Tyr185) (#4668), JNK (#9252), GAPDH(#2118), α-Tubulin (#3873) and eNOS (#32027) were from Cell Signaling Technology.

Techniques: Western Blot, Transfection, Two Tailed Test

Journal: Cancer Medicine

Article Title: Mitochondrial phosphoenolpyruvate carboxykinase promotes tumor growth in estrogen receptor‐positive breast cancer via regulation of the mTOR pathway

doi: 10.1002/cam4.4969

Figure Lengend Snippet: PCK2 promotes cell cycle progression in ER + breast cancer cells by regulating mTOR pathway. (A) The gene sets (mTORC1) was negatively enriched in MCF‐7 cells with PCK2 knockdown. (B) The expression status of downstream signals of mTORC1, S6K, and 4EBP‐1 in MCF‐7 (left) and T47D (right) cells with and without PCK2 knockdown after 48 h incubation. (C) The expression status of downstream signals of mTORC1, S6K, and 4EBP‐1 in MCF‐7 (left) and T47D (right) cells with and without PCK2 overexpression after 48 h incubation. pEZ‐LV157 is the vector control. (D) The intracellular glutamine level in MCF‐7 cells with and without PCK2 knockdown checked after 24 h incubation. (E) The expression of downstream signals of mTORC1 and cell cycle‐regulating molecules in MCF‐7 cells with and without PCK2 knockdown and with or without supplement of glutamine for 24 h. (F) The expression of downstream signals of mTORC1 and cell cycle associated molecules in MCF‐7 cells treated with indicated doses of RAD001 for 24 h.

Article Snippet: Antibodies against S6K (#9202), 4EBP‐1 (#9452), phospho‐4EBP‐1 Thr70 (#9455s), and cyclin D1 (#2926) were purchased from Cell Signaling.

Techniques: Expressing, Incubation, Over Expression, Plasmid Preparation

Journal: Cells

Article Title: The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth

doi: 10.3390/cells11071098

Figure Lengend Snippet: Measures of MPS and anabolic signaling in response to treatment with CON, NAFLD and ESLD serum. Myotubes were treated for 4 h with ex vivo serum from age-matched control (CON), non-alcoholic fatty liver disease (NAFLD) and end stage liver disease (ESLD) patients. ( a ) Representative western blot for puromycin and total protein, ( b ) representative western blots for anabolic signaling targets and loading control, ( c ) puromycin incorporation, ( d ) phospho-mTOR (Ser2448)/total-mTOR, ( e ) phospho-eEF2 (Thr56)/total-eEF2, ( f ) phospho-p70S6K (Thr389)/total-p70S6K, ( g ) phospho-AktSer473/total-Akt, ( h ) phospho-RPS6 (Ser240/244)/total-RPS6, ( i ) phospho-4EBP-1 (Thr37/46)/total-4EBP-1. Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with n = 4 per group and each data point corresponding to the average of 3 technical repeats.

Article Snippet: As a loading control, membranes were stained with Ponceau S solution and blocked with 5% milk or bovine serum albumin (BSA) diluted in Tris-buffered saline and 0.1% Tween-20 (TBST) for 1 h. Overnight, the membranes were incubated at 4 ° C with the following primary antibodies: phospho-mTOR Ser2448 (#2971, CST), total mTOR (#2972, CST), phospho-eukaryotic elongation factor 2 (eEF2) Thr56 (#2331, CST), total eEF2 (#2332, CST), phospho-ribosomal protein S6 kinase beta-1 (p70S6K1) Thr389 (#9205, CST), total p70S6K1 (#9202, CST), phospho-protein kinase B (Akt) Ser473 (#3787, CST), total Akt (#9272, CST), phospho-RPS-6 Ser240/244 (#5364, CST), total RPS-6 (#2217, CST), phospho-4EBP-1 Thr37/46 (#9459, CST), total 4EBP1 (#9452, CST), mouse IgG2a monoclonal anti-puromycin (clone 12D10, 1:5000, Merck Millipore), total OXPHOS rodent cocktail (ab110413, abcam), myostatin (ab201954, abcam), muscle RING finger 1 (MuRF-1) (sc-398608, Santa Cruz Biotechnology, Dallas, TX, USA) and muscle atrophy F-box (MAFbx) (AM3141, ECM Biosciences, Versailles, KY, USA), LC3A/B (#12741, CST) and Caspase-3 (D3R6Y) (#14220, CST).

Techniques: Ex Vivo, Western Blot