Journal: Endocrinology

Article Title: Neuropeptide Y Promotes mTORC1 to Regulate Chondrocyte Proliferation and Hypertrophy.

doi: 10.1210/endocr/bqac213

Figure Lengend Snippet: Figure 4. NPY activated mTORC1 activity in growth plate chondrocytes. (A, B) Chondrocytes were treated with 100 nM NPY for 48 hours in normal medium; expression of S6K1 phosphorylation (p-S6K1) and S6 phosphorylation (p-S6) were detected by Western blotting. (C, D) Chondrocytes were infected with LV3-NC or LV3-shNpy for 48 hours in normal medium. The expression of p-S6K1 and p-S6 were detected by western blotting. (E) Western blotting analysis of chondrocytes treated with or without 100 nM NPY. Cells were cultured in differentiation medium on day 0 and were harvested on day 4 and day 8, respectively. (F) Western blotting analysis of chondrocytes infected with LV3-NC or LV3-shNpy. A representative blot from 3 independent experiments was presented for each protein. (G) Immunohistochemistry assay of p-S6 expression in metatarsal growth plate. Higher magnification is shown below. Scale bars, 100 μm. The percentages of p-S6 positive cells were quantitatively analyzed (n = 6/group). *P < .05, **P < .01, ***P < .001. Data are presented as mean ± SD.

Article Snippet: Proteins were separated by 8% to 15% sodium dodecyl sulfate polyacryl amide gel electrophoresis; separated proteins were transferred onto PVDF membranes (Millipore) and were probed with the following primary antibodies: rabbit monoclonal antibodies against NPY (Cell Signaling Technology, 11976, RRID:AB_2716286), p-S6K1 (Cell Signaling Technology, 97596, RRID:AB_2800283), S6K1 (Cell Signaling Technology, 5707, RRID:AB_10 694087), pS6 (Cell Signaling Technology, 4858, RRID: AB_916156), S6 (Cell Signaling Technology, 2217, RRID: AB_331335), p-ERK (Cell Signaling Technology, 4370, RRID:AB_2315112), ERK (Cell Signaling Technology, 4695, RRID:AB_390779), TSC2 (Cell Signaling Technology, 4308, RRID:AB_10547134), p-TSC2 (Cell Signaling Technology, 40729, RRID:AB_2799183); mouse monoclonal antibodies against GAPDH (Santa Cruz Biotechnology, sc-365062, RRID:AB_10847862).

Techniques: Activity Assay, Expressing, Phospho-proteomics, Western Blot, Infection, Cell Culture, Immunohistochemistry

Journal: Endocrinology

Article Title: Neuropeptide Y Promotes mTORC1 to Regulate Chondrocyte Proliferation and Hypertrophy.

doi: 10.1210/endocr/bqac213

Figure Lengend Snippet: Figure 7. NPY/NPY2R stimulated mTORC1 activity by activation ERK1/2. (A, B) Chondrocytes were treated with or without 1 μM Y2R antagonist (Y2Rant) in the presence or absence of 100 nM NPY. The expression of S6K1 phosphorylation (p-S6K1) and S6 phosphorylation (p-S6) were detected by Western blotting. (C, D) Western blotting analysis of chondrocytes transfected with Npy2r siRNA in the presence or absence of 100 nM NPY. (E) Western blotting analysis of chondrocytes transfected with Npy2r plasmid in the presence or absence of 100 nM NPY. (F) The expression of ERK1/2 phosphorylation (p-ERK1/2) was detected by Western blotting in chondrocytes treated with or without 1 μM Y2Rant in the presence or absence of 100 nM NPY. (G) Western blotting analysis of chondrocytes transfected with Npy2r plasmid in the presence or absence of 100 nM NPY. (H) Chondrocytes were treated with or without 50 μM PD98059 (ERK1/2 inhibitor) in the presence or absence of 100 nM NPY. Levels of p-S6K1 and p-S6 were detected by Western blotting. (I) Western blotting analysis of p-ERK phosphorylated TSC2 at sites of Ser664 in chondrocytes. A representative blot from 3 independent experiments was presented for each protein. *P < .05, **P < .01, ***P < .001. Data are presented as mean ± SD.

Article Snippet: Proteins were separated by 8% to 15% sodium dodecyl sulfate polyacryl amide gel electrophoresis; separated proteins were transferred onto PVDF membranes (Millipore) and were probed with the following primary antibodies: rabbit monoclonal antibodies against NPY (Cell Signaling Technology, 11976, RRID:AB_2716286), p-S6K1 (Cell Signaling Technology, 97596, RRID:AB_2800283), S6K1 (Cell Signaling Technology, 5707, RRID:AB_10 694087), pS6 (Cell Signaling Technology, 4858, RRID: AB_916156), S6 (Cell Signaling Technology, 2217, RRID: AB_331335), p-ERK (Cell Signaling Technology, 4370, RRID:AB_2315112), ERK (Cell Signaling Technology, 4695, RRID:AB_390779), TSC2 (Cell Signaling Technology, 4308, RRID:AB_10547134), p-TSC2 (Cell Signaling Technology, 40729, RRID:AB_2799183); mouse monoclonal antibodies against GAPDH (Santa Cruz Biotechnology, sc-365062, RRID:AB_10847862).

Techniques: Activity Assay, Activation Assay, Expressing, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Aerobic Exercise Ameliorates Cancer Cachexia-Induced Muscle Wasting through Adiponectin Signaling

doi: 10.3390/ijms22063110

Figure Lengend Snippet: Aerobic exercise enhances the stimulation of Akt signaling cascade. Western blot analysis was performed on the TA of the mice in using anti-phospho-Akt (p-Akt) and pan-Akt (total Akt) antibodies ( A ), anti-phospho- and pan-mTOR ( C ), anti-phospho- and pan-p70S6 kinase ( E ), and anti-phospho and pan-4EBP-1 antibodies ( G ). The data represents the results of three independent experiments. Densitometric analyses of Western blot shown in ( A , C , E , G ) are presented in ( B , D , F , H ), respectively. The bar graphs indicate the mean relative expression ± SD. Statistical analysis was performed by Kruskal-Wallis analysis followed by Mann-Whitney U tests. *: p < 0.05.

Article Snippet: The following primary antibodies and dilutions were used: p-Akt (pSer473; 1:1000; Cell signaling Technology, Danvers, MA, USA, #4060), Akt (1:1000; Cell signaling technology, #9272), p-mTOR (pSer2448; 1:1000; Cell signaling technology, #5536), mTOR (1:1000; Cell signaling technology, #2972), p-p70S6K (pThr389; 1:1000; Cell signaling technology #9234), p70S6K (1:1000; Cell signaling technology #2708), p-4EBP-1 (pSer65; 1:1000; Cell Signaling Technology, #9451), 4EBP (1:1000; Cell signaling technology #9452), LC3 (1:250; rabbit polyclonal antibody raised against rat LC3B, kindly provided by Prof. Ueno, Juntendo University), β-actin (1:5000; Proteintech, Tokyo, Japan, #60008-1-Ig).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Aerobic Exercise Ameliorates Cancer Cachexia-Induced Muscle Wasting through Adiponectin Signaling

doi: 10.3390/ijms22063110

Figure Lengend Snippet: Effects of adiponectin treatment on the protein synthesis pathways in the in vitro cancer cachexia model. C2C12 myotubes were treated with C26 derived conditioned medium (C26-CM) or 1 µg/mL adiponectin, as indicated in . Total proteins were analyzed by western blotting using antibodies for phospho-Akt and pan-Akt ( A ), phospho-mTOR and pan-mTOR ( C ), and phospho- and pan-p70S6 kinase (S6K) ( E ). A representative result from one of three independent experiments is shown. Densitometric analyses of western blotting shown in ( A , C , E ) are presented in ( B , D , F ) respectively. The bar graphs indicate the mean relative expression ± SD. Statistical analysis was performed by Kruskal-Wallis analysis followed by Mann-Whitney U tests. *: p < 0.05.

Article Snippet: The following primary antibodies and dilutions were used: p-Akt (pSer473; 1:1000; Cell signaling Technology, Danvers, MA, USA, #4060), Akt (1:1000; Cell signaling technology, #9272), p-mTOR (pSer2448; 1:1000; Cell signaling technology, #5536), mTOR (1:1000; Cell signaling technology, #2972), p-p70S6K (pThr389; 1:1000; Cell signaling technology #9234), p70S6K (1:1000; Cell signaling technology #2708), p-4EBP-1 (pSer65; 1:1000; Cell Signaling Technology, #9451), 4EBP (1:1000; Cell signaling technology #9452), LC3 (1:250; rabbit polyclonal antibody raised against rat LC3B, kindly provided by Prof. Ueno, Juntendo University), β-actin (1:5000; Proteintech, Tokyo, Japan, #60008-1-Ig).

Techniques: In Vitro, Derivative Assay, Western Blot, Expressing, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans .

doi: 10.3390/ijms24010612

Figure Lengend Snippet: Figure 6. Expression of idha-1 negatively modulates the phosphorylated S6K levels. The level of phosphorylated S6K (p-S6K), a TOR activity indicator, in each strain was determined and normal- ized with β-actin level. (a,b) Long-lived idha-1 overexpression strain (red bar, idha-1 o/e) displays significantly reduced p-S6K levels compared with the control (grey bar) (** p < 0.01, t test). Each bar represents mean ± SD, n = 4. (c,d) Short-lived idha-1 knockdown strains (blue bar, N2 idha-1 (RNAi) 5′; and yellow bar, N2 idha-1 (RNAi) 3′) exhibit significantly increased p-S6K levels compared with the control (black bar, N2 EV) (* p < 0.05, t test). Each bar represents mean ± SD, n = 3.

Article Snippet: The primary antibodies used were listed below: IDHA-3A (Genetex, GTX114486), beta-actin (GeneTex, GTX109639), and p-S6K (Cell Signaling, Cat# 9209).

Techniques: Expressing, Activity Assay, Over Expression, Control, Knockdown

Journal: Cancer Research

Article Title: Sorafenib Enhances Pemetrexed Cytotoxicity through an Autophagy-Dependent Mechanism in Cancer Cells

doi: 10.1158/0008-5472.can-11-0898

Figure Lengend Snippet: Figure 4. Knockdown of PDGFR-b, mTOR, or p70 S6K enhances pemetrexed toxicity and inhibition of ERK1/2 suppresses drug combination toxicity. A, tumor cells, as indicated, growing in log phase were isolated 24 hours after plating and subjected to SDS-PAGE and immunoblotting against the indicated proteins as described in the Materials and Methods section. The intensity of immunostaining was normalized to either GAPDH for proteins or for phosphoproteins to the equivalent dephosphorylated protein; these values were then normalized with the intensity value of each protein/phospho-protein in BT474 cells defined as 1.00 ( SEM; n ¼ 3; #, P < 0.05 greater than value in BT474; @, P < 0.05 greater than value in parental MCF7 and BT474; þ, P < 0.05

Article Snippet: Plasmids to express active p70 S6K and active mTOR were purchased from Addgene.

Techniques: Knockdown, Inhibition, Isolation, SDS Page, Western Blot, Immunostaining

Journal: Frontiers in Nutrition

Article Title: Maternal docosahexaenoic acid supplementation during lactation improves exercise performance, enhances intestinal glucose absorption and modulates gut microbiota in weaning offspring mice

doi: 10.3389/fnut.2024.1423576

Figure Lengend Snippet: Effect of maternal DHA supplementation during lactation on the mTOR signaling pathway in weaning mice. (A) Representative Western blots of p-mTOR (S2448), total mTOR, p-p70S6 kinase (T389), total p70S6 kinase, p-S6 ribosomal protein (S240/244) and total S6 ribosomal protein, and (B, C, D) the relative band intensities of phosphorylation levels normalized to their respective total proteins in jejunal tissue of mice from different groups. Data were expressed as the mean ± SEM; n = 4 per group. Significance was set at p < 0.05 ( * p < 0.05, ** p < 0.01). L-DHA: 150 mg/(kg body weight day) DHA; H-DHA: 450 mg/(kg body weight day) DHA. DHA, docosahexaenoic acid; mTOR, mammalian target of rapamycin.

Article Snippet: After transfer, the PVDF membrane was blocked for 1 h in 5% fat-free milk and then was, respectively, incubated overnight at 4°C with dilutions of primary antibodies: GLUT2 (A12307, 1:2000, Abclonal, China), SGLT1 (bs-1128R, 1:2000, Bioss, China), S6 ribosomal protein (abs131865, 1:2000, Absin, China), p-Ser240/244-S6 ribosomal protein (ab215214, 1:2000, Abcam, UK), p70S6 kinase (14485-1-AP, 1,1,000, Proteintech, China), p-Thr389-p70S6 kinase (9,234, 1,1,000, Cell Signaling Technology, United States), mammalian target of rapamycin (mTOR, T55306, 1:2000, Abmart, China), p-S2448-mTOR (5,536, 1,1,000, Cell Signaling Technology), β-actin (T40104, 1:4000, Abmart).

Techniques: Western Blot, Phospho-proteomics

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 , b HAEC were treated with 1 nM rapa for 1 h prior to western blot analysis and quantification of eNOS protein ( n = 3, a ) as well as the ratio of phosphorylated Ser1177 or Thr495 to total eNOS ( b ). c HAEC were transfected with siRNA followed by western blotting and quantification of eNOS expression ( n = 3). d , e HAEC were treated with 1 nM rapa for 1 h ( d ) or transfected with siRNA prior to quantitative PCR ( n = 5–7). f At 48 h, post transfection with siRNA against RPS6KB1 , HAEC were submitted to western blot analysis, quantification of eNOS expression ( n = 3), and real-time PCR ( n = 6). g HAEC were transfected with pcDNA3.1 (sham) or pcDNA3.1 EIF4EBP-1 plasmids followed by western blot analysis, quantification of eNOS expression ( n = 3) and quantitative PCR ( n = 6). Error bars correspond to standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. ctrl or sictrl or sham; one sample t test ( a , b , d , f , g ) or repeated measures (RM) one-way ANOVA with Dunnett’s test ( c , e ).

Article Snippet: Negative control siRNA (sc-37007), siRNA targeting KLF2 (sc-35818), RPTOR (encoding Raptor) (sc-44069), RICTOR (sc-61478), MTOR (sc-35409), RPS6KB1 (encoding p70S6K) (sc-36165) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pcDNA3.1 EIF4EBP1 (encoding 4EBP-1) was constructed using PCR with primers containing restriction enzyme sites. siRNA or plasmid was transfected into HAEC using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturers’ instructions.

Techniques: Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction

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 , b HAEC were treated with 1 nM rapa or transfected with siRNA prior to Western blot analysis and quantification of KLF2 expression ( n = 3–4). c , d HAEC were transfected with KLF2 -targeting siRNA followed by Western blotting (c) and quantitative PCR ( n = 4, d ). e At 48 h post-transfection with siRNA against KLF2 , relative NO level of the culture medium was measured with the colorimetric assay ( n = 5). f HAEC were treated with 1 nM rapa for 1 h, prior to quantitative PCR to measure mRNA ( n = 3). g , h HAEC were transfected with siRNA against RPS6KB1 followed by quantitative PCR ( n = 7, g) or western blotting ( n = 4, h ) to quantify KLF2 expression. ** p < 0.01; *** p < 0.001 vs. sictrl or ctrl; one-sample t test ( a , d , f , g , h ) or RM one-way ANOVA with Dunnett’s test ( b ) or unpaired two-tailed t test ( e ).

Article Snippet: Negative control siRNA (sc-37007), siRNA targeting KLF2 (sc-35818), RPTOR (encoding Raptor) (sc-44069), RICTOR (sc-61478), MTOR (sc-35409), RPS6KB1 (encoding p70S6K) (sc-36165) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pcDNA3.1 EIF4EBP1 (encoding 4EBP-1) was constructed using PCR with primers containing restriction enzyme sites. siRNA or plasmid was transfected into HAEC using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturers’ instructions.

Techniques: Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Colorimetric Assay, Two Tailed Test