Journal: The Journal of nutrition

Article Title: Dairy and Dairy Alternative Supplementation Increase Integrated Myofibrillar Protein Synthesis Rates, and Are Further Increased when Combined with Walking in Healthy Older Women.

doi: 10.1093/jn/nxab358

Figure Lengend Snippet: FIGURE 5 Total protein content of canonical anabolic signaling factors in healthy older women. Box and whisker plots showing the relative protein content of mTOR, p70S6K, rpS6, and 4E-BP1. The horizontal line within the boxes depicts of the median of each group, and the cross the mean. Upper and lower edges of the box represent the 75th and 25th quartiles, respectively. Upper and lower whiskers represent the maximum and minimum values, respectively. Data were analyzed using a linear mixed-effects model. Data that do not share a common letter are significantly different from each other (P ≤0.05). Insets above each box and whisker plot show representative blots at each phase of the study for 1 participant, as well as the associated standard curve from the same gel (5, 10, and 15 μg of protein loaded). Panel E shows a representative membrane image taken after proteins were transferred from gel to membrane and panel F shows representative images of each time point and calibration curve standards. Sample sizes: AB (BL, n = 8; P1, n = 8; P2, n = 7; P3, n = 7), SM (BL, n = 7; P1, n = 6; P2, n = 7; P3, n = 6), WM (BL, n = 7; P1, n = 7; P2, n = 6; P3, n = 7). AB, almond beverage; BL, baseline (day 0); P1, End of Phase 1 (day 4); P2, End of Phase 2 (day 7); P3, End of Phase 3 (day 10); mTOR, mechanistic target of rapamycin; p70S6K, p70 ribosomal protein S6 kinase; rpS6, ribosomal protein S6; 4E-BP1, Eukaryotic translation initiation factor 4E-binding protein 1; RAU, relative arbitrary units; SM, skim milk; WM, whole milk.

Article Snippet: Primary antibodies against mechanistic target of rapamycin (mTOR) (1:1000, 7C10, Cat #2983S), p70S6K (1:1000, 49D7, Cat #2708S), rpS6 (1:1000, 5G10, Cat #2217S), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) (1:1000, 53H11, Cat# 9644S) and horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000, Cat #7074S) were purchased from Cell Signaling Technologies.

Techniques: Whisker Assay, Membrane, Binding Assay

Journal: Molecular Metabolism

Article Title: An amino acid-defined diet impairs tumour growth in mice by promoting endoplasmic reticulum stress and mTOR inhibition

doi: 10.1016/j.molmet.2022.101478

Figure Lengend Snippet: Glu supplementation blunts the effects of EAAm on ATF4 induction, mTOR inhibition and cell growth . (A) Western blot analysis of nuclear extracts (ATF4) or total lysates (p70S6K) of M14 untreated (CTRL), incubated with EAAm mixture, or incubated with EAAm mixture plus Glu, Gly, Asp or Ala 10 mM for 3 h. (B) Clonogenic assay in M14 cells left untreated (CTRL), incubated with EAAm mixture (EAAm), or incubated with EAAm mixture plus Glu 10 mM (EAAm + GLU) for 24 h (C) sestrin2 (SESN2) protein expression in HeLa cells transfected with non-targeting (scrambled) or SESN2- specific siRNA. Vinculin is shown as a loading control (D). p-S6 levels immunoblot analysis in Hela cells transfected with control non-targeting siRNA (scrambled), or siRNA against sestrin2 (SESN2), and treated (+) or not (−) with EAAm 1%.for 3 h (E) MTT assay of Hela cells transfected with non-targeting (scrambled) siRNA or siRNA against sestrin 2 (SESN2) and incubated with culture medium (CTRL) or supplemented with 1% EAAm for 24 h (EAAm). Mean (n = 4) ± SEM, ∗∗ P < 0.01 vs. CTRL. Two-way ANOVA followed by Tukey's post hoc test.

Article Snippet: Antibodies used were: ATF4 (Santa Cruz Biotechnology cat # sc-200), p-70S6K (Thr389) (Cell Signaling cat # 9205), p70S6K (Cell Signaling cat # 9202), Phospho-S6 Ribosomal Protein (Ser235/236) (Cell Signaling cat # 4858) S6 Ribosomal Protein (Cell Signaling cat # 2217) Menin (Active Motifs cat # 2615023), CytC (Cell Signaling cat # 4280), COXIV (Cell Signaling cat # 4844), PARP (Trevigen # 4338), Caspase 3 (Cell Signaling cat # 9662), HIF-1α (BD Biosciences cat # 610958), β-Actin (Cell Signaling cat # 4970), p-eIF2α (Ser51) (Cell Signaling cat # 3398), eIF2α (Cell Signaling cat # 5324), PP2CM (Abcam cat # 135286), p-BCKDHA (Ser293) (Abcam cat # 200577), BCKDHA (Abcam cat # 138460), GAPDH (Cell Signaling cat # 2118), SESN-2 (Santa Cruz Biotechnology # sc-101249), BCKDK (Santa Cruz Biotechnology # sc-374425).

Techniques: Inhibition, Western Blot, Incubation, Clonogenic Assay, Expressing, Transfection, Control, MTT Assay

Journal: Molecular Metabolism

Article Title: An amino acid-defined diet impairs tumour growth in mice by promoting endoplasmic reticulum stress and mTOR inhibition

doi: 10.1016/j.molmet.2022.101478

Figure Lengend Snippet: EAAm activates BCAA catabolism, and BT2 potentiates the effects of EAAm (A) Expression of PP2Cm and phosphorylation levels of BCKDHA dehydrogenase in Hela cells left untreated (CTRL) or treated with or EAAm (EAAm) for 3 h (B) phospho-BCKDHA dehydrogenase, phospho-S6 ribosomal protein and nuclear expression of ATF4 in HeLa (left) and M14 (right) cells treated with vehicle (CTRL) or with the BCKDK inhibitor BT2 (100 μM) for 5 h. Menin is shown as a nuclear loading control for ATF4. (C) Glycolysis rate (ECAR) in M14 cells vehicle-treated (CTRL) or treated with BT2 200 μM for 1 h (D) MTT assay in M14 cells treated with vehicle (CTRL) or treated with BT2 or BT2 plus EAAm for 16 h with the indicated doses. Mean (n = 4) ± SEM from at least two experiments. ∗ P < 0.05 and ∗∗∗ P < 0.001 vs. CTRL, ### P < 0.001 vs. BT2, §§§ P < 0.001 vs. EAAm. Two-way ANOVA followed by Tukey's post hoc test.

Article Snippet: Antibodies used were: ATF4 (Santa Cruz Biotechnology cat # sc-200), p-70S6K (Thr389) (Cell Signaling cat # 9205), p70S6K (Cell Signaling cat # 9202), Phospho-S6 Ribosomal Protein (Ser235/236) (Cell Signaling cat # 4858) S6 Ribosomal Protein (Cell Signaling cat # 2217) Menin (Active Motifs cat # 2615023), CytC (Cell Signaling cat # 4280), COXIV (Cell Signaling cat # 4844), PARP (Trevigen # 4338), Caspase 3 (Cell Signaling cat # 9662), HIF-1α (BD Biosciences cat # 610958), β-Actin (Cell Signaling cat # 4970), p-eIF2α (Ser51) (Cell Signaling cat # 3398), eIF2α (Cell Signaling cat # 5324), PP2CM (Abcam cat # 135286), p-BCKDHA (Ser293) (Abcam cat # 200577), BCKDHA (Abcam cat # 138460), GAPDH (Cell Signaling cat # 2118), SESN-2 (Santa Cruz Biotechnology # sc-101249), BCKDK (Santa Cruz Biotechnology # sc-374425).

Techniques: Expressing, Phospho-proteomics, Control, MTT Assay

Journal: Molecular Pain

Article Title: Bortezomib and metformin opposingly regulate the expression of hypoxia-inducible factor alpha and the consequent development of chemotherapy-induced painful peripheral neuropathy

doi: 10.1177/1744806919850043

Figure Lengend Snippet: (a) Treatment of DRG cultures with metformin (20 mM) reduced the levels of HIF1A, activated AMPK which is confirmed by increased phosphorylation of Thr172 and suppressed translation which is validated by reduction in the phosphorylation of rS6 (HIF1A, * P = 0.0119; AMPK, ** P = 0.0046; rS6, * P = 0.0148; six wells/group). (b) Co-injection of metformin (Met, 150 mg/kg) with bortezomib (Bort) for five consecutive days prevented the development of bortezomib-induced neuropathic pain (**** P <0.0001, five mice/group). HIF1A: hypoxia-inducible factor 1 alpha; AMPK: AMP-activated protein kinase.

Article Snippet: Primary antibodies include HIF1A, p-AMPK, AMPK, p-rS6, and rS6 (1:1000 Cell Signaling Technology, Cat # 14179, 2535, 2603, 2215, 2217) and beta-III-tubulin (1:50,000 Promega, Cat # G7121).

Techniques: Phospho-proteomics, Injection