Journal: The Journal of Physiology

Article Title: Intermittent time‐restricted eating may increase autophagic flux in humans: an exploratory analysis

doi: 10.1113/jp287938

Figure Lengend Snippet: Figure 2. Validation of the autophagy measurement methodology A, MAP1LC3B was knocked out using CRISPR-Cas9 gene editing. A western blot for LC3B shows complete loss of LC3B-I and LC3B-II bands around 15 kDa. B, quantification of three independent cell lysates from wild-type and LC3B KO cells without and with CQ on the same ELISA that was used for the main analysis of this study shows very high specificity for the target protein. An unpaired t test was used to test significance. C, three individual vacutainers containing blood were taken from a participant and processed in parallel. Resulting cell pellets were saponin washed in parallel and both the saponin-washed pellet (P: containing lipid-bound proteins such as lipidated LC3B-II) and the saponin wash supernatant (S: containing soluble cytosolic proteins such as non-lipidated LC3B-I) are shown. Western blots for LC3B show LC3B-II that generally runs just below 15 kDa in the pellet, and LC3B-I that generally runs just above 15 kDa in the supernatant. Total protein staining is also shown to illustrate the consistency of the saponin extraction and the contrasting protein content of the saponin-soluble (S) and saponin-insoluble (P) fractions. D, three different blood tubes were taken from an individual and processed in parallel. Resulting cell pellets were processed by ELISA. LC3B in saponin-washed cell pellets (three tubes from five individuals, with or without CQ) were quantified by ELISA and grouped appropriately. Addition of CQ causes increases in LC3B-II. Significance was quantified using multiple t tests and P-values are adjusted for multiple measures. Bars = mean ± SD for three parallel processed samples for each participant. Parallel samples appear as datapoints around the bars. E, variation for each participant presented in D, with or without CQ, is displayed as the coefficient of variation (CV).

Article Snippet: To determine LC3B-II sample concentration, 5 μg per well (diluted in CST FastScan 1× Cell Extraction Buffer) of sample was loaded in triplicate on a FastScan Total LC3B ELISA Kit (Cell Signaling Technology, 35172).

Techniques: Biomarker Discovery, CRISPR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Extraction

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) Enzyme-linked immunosorbent assay (ELISA assay) showed that the platelet autophagy biomarkers in cirrhotic patients were significantly reduced compared to the healthy controls. (B–C) The levels of autophagy biomarkers negatively correlated with the Child-Pugh score and the severity of TP. (D) Transmission electron microscopy showed that TP patients had significantly reduced platelet autophagosomes compared to healthy controls. (E) The serum endogenous H 2 S levels in TP patients were significantly reduced compared to healthy controls. (F) Spearman correlation analysis indicated that serum H 2 S levels positively correlated with platelet autophagy markers LC3 and ATG7 and negatively correlated with SQSTM1 in TP patients. * indicates p <0.05. Scale bar: 1 µm.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) NaHS increased the relative LC3-II expression and attenuated the SQSTM1 expression dose-dependently. (B) Compared with the Baf A1 treatment alone, the LC3-II expression was significantly increased after NaHS+Baf A1 treatment indicated that the platelet autophagosome synthesis was increased by H 2 S. * indicates p <0.05. “+” indicates using Bafilomycin (Baf) A1 treatment, “−” indicates using culture medium treatment. NC, negative control.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Expressing, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) Establishment of mice model of cirrhotic thrombocytopenia. (B) NaHS injection increased the platelet LC3-II expression and reduced the SQSTM1 expression in a dose-dependent manner. After injection with hydroxocobalamin (HC, H 2 S scavenger), the platelet LC3-II expression was significantly reduced, while the SQSTM1 expression was significantly increased. (C) After injection of different concentrations of NaHS or HC, there was no significant change in the platelet count of the mice at an indicated time. (D) After the injection of different concentrations of NaHS, the platelet aggregation rate significantly decreased on Day 15. * indicates p <0.05. i.p, intraperitoneal injection; biw, twice a week; NS, 0.9% normal saline; MPA, microscopic polyangiitis.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Injection, Expressing, Saline

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) Enzyme-linked immunosorbent assay (ELISA assay) showed that the platelet autophagy biomarkers in cirrhotic patients were significantly reduced compared to the healthy controls. (B–C) The levels of autophagy biomarkers negatively correlated with the Child-Pugh score and the severity of TP. (D) Transmission electron microscopy showed that TP patients had significantly reduced platelet autophagosomes compared to healthy controls. (E) The serum endogenous H 2 S levels in TP patients were significantly reduced compared to healthy controls. (F) Spearman correlation analysis indicated that serum H 2 S levels positively correlated with platelet autophagy markers LC3 and ATG7 and negatively correlated with SQSTM1 in TP patients. * indicates p <0.05. Scale bar: 1 µm.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Berberine Improves Cancer-Derived Myocardial Impairment in Experimental Cachexia Models by Targeting High-Mobility Group Box-1

doi: 10.3390/ijms25094735

Figure Lengend Snippet: Effects of BBR on myocardial damage in a rat cancer cachexia model ( A ) Experimental protocol. F344 male rats (5 weeks old, 5 rats each group, SLC Japan, Shizuoka, Japan) were inoculated with RCN9 cancer cells (1 × 10 7 ) intraperitoneally with or without administration of BBR (48 μg/mL, free drink). ( B ) Body weight. ( C ) Tumor weight. ( D ) Ascites volume. ( E ) Cytokine concentration in the ascites fluid. ( F ) Cardiac weight. ( G ) Myocardial maturity (SDS-MYL1). ( H ) Myocardial ROS (4HNE). ( I ) Myocardial cell death (serum Troponin T levels). ( J ) Autophagy (LC3B). ( K ) Cardiac function (serum BNP). Statistical significance was calculated using an ordinary ANOVA. N, no tumor; C, control; BBR, berberine; HMGB1, high-mobility group box-1; TNF, tumor necrosis factor; SDS-MYL1, sodium dodecyl sulfate-soluble myosin light chain-1; 4HNE, 4-hydroxynonenal; LC3, microtubule-associated protein 1A/1B-light chain 3-B; BNP, brain natriuretic peptide.

Article Snippet: LC3B , #35172 , Cell Signaling, Danvers, MA, USA , .

Techniques: Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Berberine Improves Cancer-Derived Myocardial Impairment in Experimental Cachexia Models by Targeting High-Mobility Group Box-1

doi: 10.3390/ijms25094735

Figure Lengend Snippet: Primer sets, antibodies, and ELISA kits.

Article Snippet: LC3B , #35172 , Cell Signaling, Danvers, MA, USA , .

Techniques: Enzyme-linked Immunosorbent Assay