Journal: Oxidative Medicine and Cellular Longevity

Article Title: Recombinant Human Growth Hormone Inhibits Lipotoxicity, Oxidative Stress, and Apoptosis in a Mouse Model of Diabetic Cardiomyopathy

doi: 10.1155/2021/3899356

Figure Lengend Snippet: rhGH improved lipid metabolism in db/db mice. (a) BODIPY-stained (green fluorescence) and DAPI-stained (blue fluorescence) photomicrographs and PAS staining in the cardiac tissue. (b) Cardiac p-AMPK, CPT-1, PGC1-ɑ, PPAR- α , CD36, and SCD-1 levels were measured by western blotting. (c) Relative protein expression was quantified. (d) Representative immunohistochemistry for p-AMPK, CPT-1, PGC1-ɑ, PPAR- α , CD36, and SCD-1 in the cardiac tissues. (e) Positive expression was quantified. Scale bar: 100 μ m. The arrows indicate positively stained cells. n = 3 per group. ∗ P < 0.05 vs. db/db+rhGH group, ∗∗ P < 0.01 vs. db/db+rhGH group, & P < 0.05 vs. db/db group, and # P < 0.01 vs. db/db group.

Article Snippet: The primary antibodies were against ANP (rabbit anti-ANP antibody, 1 : 500; Invitrogen, USA), BNP (rabbit anti-BNP antibody, 1 : 500; Invitrogen), CD36 (rabbit anti-CD36 antibody, 1 : 1000; Proteintech), SCD-1 (rabbit anti-SCD-1 antibody, 1 : 1000; Abcam), PGC1-ɑ (rabbit anti-PGC1-ɑ antibody, 1 : 1000; Proteintech), CPT-1 (rabbit anti-CPT-1 antibody, 1 : 1000; Proteintech), p-AMPK (rabbit anti-p-AMPK antibody, 1 : 1000; Proteintech), NRF2 (rabbit anti-NRF2 antibody, 1 : 500; Proteintech), HO-1 (rabbit anti-HO-1 antibody, 1 : 1000; Abcam), PPAR- α (rabbit anti-PPAR- α antibody, 1 : 500; Proteintech), SOD (rabbit anti-SOD antibody, 1 : 2000; Proteintech), NOX4 (rabbit anti-NOX4 antibody, 1 : 1000; Proteintech), CAT (rabbit anti-CAT antibody, 1 : 1000; Proteintech), BAX (rabbit anti-BAX antibody, 1 : 5000; Proteintech), Bcl-2 (rabbit anti-Bcl-2 antibody, 1 : 2000; Proteintech), cleaved caspase-3 (rabbit anti-cleaved caspase-3 antibody, 1 : 1000; Cell Signaling Technology), t-caspase-3 (rabbit anti-t-caspase-3 antibody, 1 : 1000; Proteintech), and anti- β -actin (1 : 1000; Proteintech).

Techniques: Staining, Fluorescence, Western Blot, Expressing, Immunohistochemistry

Journal: Autophagy

Article Title: Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

doi: 10.1080/15548627.2021.1972403

Figure Lengend Snippet: Figure 2. Enhanced PRKAA1-dependent autophagy-mediated proteolysis in Mem from EC after cell activation. Mem from EC, ART, and HIVneg were polyclonally activated for 6 h. (A) Levels of total and p-Thr172 PRKAA1 were determined on non-activated (NA) and polyclonally activated (PA) Mem by western blotting. i. Representative blots for all study groups. ii. Densitometric quantification of 6 independent experiments was performed using ImageQuant software (mean ± SD). Results shown represent the relative levels of p-Thr172 in PRKAA1 and were determined as follows: values for p-Thr172 PRKAA1/values in % of total PRKAA1. (B) i. Autophagy-dependent proteolysis determined in purified Mem form EC, ART and HIVneg with or without PRKAA1 inhibition (using compound C [comp. C]), and after 6 h of polyclonal activation (PA) or not (NA). ii. Confirmation of AMPK inhibition by compound C was determined by western blotting using polyclonally activated Mem from EC (representative blots of 3 independent confirmations). N = 6 for all experiments. The error bars indicate standard deviations from the means. β, symbol used for paired t-test (comparison between treated Mem and untreated control). *, symbol used for Mann-Whitney test (comparison between study groups).

Article Snippet: The primary antibodies against FOXO3 (Cell Signaling Technology, 12829S), total PRKAA1 (Cell Signaling Technology, 2532S) and p-Thr172 PRKAA1 (Cell Signaling Technology, 2531S), and ACTB/β-actin (Cell Signaling Technology, 4967S) used in western blots came from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Software, Purification, Inhibition, Comparison, Control, MANN-WHITNEY

Journal: Autophagy

Article Title: Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

doi: 10.1080/15548627.2021.1972403

Figure Lengend Snippet: Figure 3. Autophagy-mediated proteolysis is required for optimal IL21 production in Mem from EC. Autophagy-mediated proteolysis and IL21 production were both determined in activated Mem under specific BECN1 gene silencing (siRNA IDs: 137,198), or PRKAA1 (compound C), lysosomal (BaF or Chloro.), and protease (E64d- PepA) inactivation. (A) Levels of BECN1 were determined on Mem after 24 h of cell transfection with negative or BECN1 siRNAs by flow cytometry. i. Representative histograms of BECN1 expression in transfected Mem from EC. ii. Percentages of BECN1+ cells in Mem with or without specific BECN1 silencing for all study groups. % of BECN1 decrease was also indicated in bold for the EC’ and HIVneg’s groups. (B) Autophagy-mediated proteolysis assessed in polyclonally activated Mem with or without specific BECN1 gene silencing or chemical inhibitors. Levels of IL21 production in Mem that have then been either (C) i. polyclonally or (D) HIV-1-specifically activated for 6 h with or without specific BECN1 gene silencing or chemical inhibitors. (C) ii. Representative histograms of IL21 expression in polyclonally-activated Mem from EC with or without autophagy blockade. iii. Confirmation of PRKAA1 inhibition by compound C was determined by western blotting using polyclonally activated Mem from EC (representative blots of 3 independent confirmations). N = 6. The error bars indicate standard deviations from the means. β, symbol used for paired t-test (comparison between treated Mem and untreated control). *, symbol used for Mann-Whitney test (comparison between study groups).

Article Snippet: The primary antibodies against FOXO3 (Cell Signaling Technology, 12829S), total PRKAA1 (Cell Signaling Technology, 2532S) and p-Thr172 PRKAA1 (Cell Signaling Technology, 2531S), and ACTB/β-actin (Cell Signaling Technology, 4967S) used in western blots came from Cell Signaling Technology.

Techniques: Transfection, Flow Cytometry, Expressing, Inhibition, Western Blot, Comparison, Control, MANN-WHITNEY

Journal: Autophagy

Article Title: Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

doi: 10.1080/15548627.2021.1972403

Figure Lengend Snippet: Figure 5. Mem from EC rather use autophagy-mediated proteolysis to support the release of free glutamine. (A-D) Intracellular levels of (A) total converted glutamine and (B,C) glutamate only in Mem at 6 h of cell activation. (A) Levels of total converted glutamine within Mem from EC, which have been or not polyclonally activated with or without PRKAA1-dependent autophagy-mediated proteolysis blockade (BaF, E64d-PepA, or compound C). (B) Validation of the glutamate bioluminescence- based measurement by using Mem from HIVneg that have been treated with BPTES to block any glutamate conversion during their cell activation. (C) Levels of glutamate in polyclonally activated Mem for all study groups with or without PRKAA1-dependent autophagy-mediated proteolysis blockade (BaF, E64d-PepA, or compound C). (D) Chromatograms of extracted ion counts representing area under the curve quantification of glutamate and glutamine in polyclonally activated Mem in all study groups. (E) Chromatograms of extracted ion counts representing area under the curve quantification of glutamate and glutamine in non-activated (NA) Mem in all study groups. (F) Relative expression of intracellular glutamine and glutamate in activated Mem in all study groups. Of note, polyclonally activated Mem from EC and HIVneg counts required 10x dilution to avoid saturating signals. N = 6. β, symbol used for paired t-test (comparison between treated Mem and untreated control). *, symbol used for Mann-Whitney test (comparison between study groups).

Article Snippet: The primary antibodies against FOXO3 (Cell Signaling Technology, 12829S), total PRKAA1 (Cell Signaling Technology, 2532S) and p-Thr172 PRKAA1 (Cell Signaling Technology, 2531S), and ACTB/β-actin (Cell Signaling Technology, 4967S) used in western blots came from Cell Signaling Technology.

Techniques: Activation Assay, Biomarker Discovery, Blocking Assay, Expressing, Comparison, Control, MANN-WHITNEY

Journal: Autophagy

Article Title: Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

doi: 10.1080/15548627.2021.1972403

Figure Lengend Snippet: Figure 6. Blocking autophagy-mediated proteolysis or glutaminolysis in EC inhibits their cellular rates of mitochondrial β-oxidation. We polyclonally activated Mem from EC in the presence or absence of PRKAA1-dependent autophagy-mediated proteolysis (BaF, E64d-PepA, and compound C) or glutaminolysis (BPTES, and R162) blockade before assessing their mitochondrial respiration. (A) Representative respiratory kinetics of Mem from EC at 6 h post-polyclonal activation when cells have been treated with or without chemical inhibitors. OCR, oxygen consumption rate. (B) SRC and (C) ATP-linked respiration were determined for all culture conditions. N = 6. The error bars indicate standard deviations from the means. β, symbol used for paired t-test (comparison between treated Mem and untreated control).

Article Snippet: The primary antibodies against FOXO3 (Cell Signaling Technology, 12829S), total PRKAA1 (Cell Signaling Technology, 2532S) and p-Thr172 PRKAA1 (Cell Signaling Technology, 2531S), and ACTB/β-actin (Cell Signaling Technology, 4967S) used in western blots came from Cell Signaling Technology.

Techniques: Blocking Assay, Activation Assay, Comparison, Control

Journal: Autophagy

Article Title: Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

doi: 10.1080/15548627.2021.1972403

Figure Lengend Snippet: Figure 9. Triggering the PRKAA1 with AICAR enhances autophagy-mediated proteolysis and glutamine/glutamate availability in ART. (A) Autophagy-mediated proteolysis assessed in polyclonally activated Mem for all study groups with or without the PRKAA1 activator AICAR. We also co-cultured Mem with AICAR and protease inhibitors E64d-PepA. (B) Levels of total glutamine/glutamate were determined within Mem from ART, which have been or not polyclonally activated with or without AICAR and AICAR + E64d-PepA (co-)treatments. (C) Chromatograms of extracted ion counts representing area under the curve quantification of glutamate and glutamine in polyclonally activated or not Mem, with/without AICAR or AICAR + E64d-PepA in ART patients. Of note, polyclonally activated Mem treated with AICAR only counts required 10x dilution to avoid saturating signals. N = 6. The error bars indicate standard deviations from the means. β, symbol used for paired t-test (comparison between treated Mem and untreated control). *, symbol used for Mann-Whitney test (comparison between study groups).

Article Snippet: The primary antibodies against FOXO3 (Cell Signaling Technology, 12829S), total PRKAA1 (Cell Signaling Technology, 2532S) and p-Thr172 PRKAA1 (Cell Signaling Technology, 2531S), and ACTB/β-actin (Cell Signaling Technology, 4967S) used in western blots came from Cell Signaling Technology.

Techniques: Cell Culture, Comparison, Control, MANN-WHITNEY

Journal: Autophagy

Article Title: Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

doi: 10.1080/15548627.2021.1972403

Figure Lengend Snippet: Figure 10. Triggering the PRKAA1-dependent autophagy-mediated proteolysis with AICAR in ART improves their cellular rates of mitochondrial β-oxidation and Mem-related IL21 production. (A-C) We polyclonally activated Mem from ART in the presence or absence of PRKAA1-dependent autophagy-mediated proteolysis (AICAR with or without BaF, and E64d-PepA) induction, before assessing their mitochondrial respiration. (A) Representative respiratory kinetics of Mem from ART at 6 h post-polyclonal activation when cells have been treated with or without PRKAA1-dependent autophagy-mediated proteolysis. OCR, oxygen consumption rate. (B) SRC and (C) ATP-linked respiration were determined for all culture conditions. (D) At 6 h of polyclonal and HIV-1-specific activation, we also monitored the intracellular levels of IL21 with or without AICAR and E64d-PepA treatments. N = 6. The error bars indicate standard deviations from the means. β, symbol used for paired t-test (comparison between treated Mem and untreated control). *, symbol used for Mann-Whitney test (comparison between study groups).

Article Snippet: The primary antibodies against FOXO3 (Cell Signaling Technology, 12829S), total PRKAA1 (Cell Signaling Technology, 2532S) and p-Thr172 PRKAA1 (Cell Signaling Technology, 2531S), and ACTB/β-actin (Cell Signaling Technology, 4967S) used in western blots came from Cell Signaling Technology.

Techniques: Activation Assay, Comparison, Control, MANN-WHITNEY