rabbit polyclonal antibody against atg5  (Novus Biologicals)

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Pioglitazone Reduces Hepatic Alpha-1 Antitrypsin Accumulation Through Autophagy and AMPK Activation in Alpha-1 Antitrypsin Deficient Mice

doi: 10.1152/ajpgi.00272.2025

Figure Lengend Snippet: (A) Western blot analysis of autophagy-related proteins in HuhZ cells treated with pioglitazone (10 μM, 48 h), with or without Bafilomycin A1 (BafA1, 100 μM, 6 h). Protein levels of ATG5, p62, and LC3B were assessed. GAPDH serves as a loading control. (B) Quantification of p62 and LC3B-II band intensities from (A), normalized to GAPDH. Bars represent mean ± SD from three independent experiments. (C) Immunofluorescence staining of LC3B (green) in HuhZ cells treated with pioglitazone ± BafA1. Nuclei were counterstained with DAPI (blue). Insets show enlarged views of LC3B-positive puncta. (D) Western blot analysis of AMPK-mTOR signaling components in control and pioglitazone-treated HuhZ cells. Phosphorylation of AMPK, ULK1, and mTOR was assessed. (E) Quantification of p-AMPK, p-ULK1, and p-mTOR band intensities from (D), normalized to GAPDH. Bars show mean ± SD. (F) Immunofluorescence staining of p-AMPK (green) and actin (red) in control and pioglitazone-treated HuhZ cells. Nuclei were counterstained with DAPI (blue). Merged images show increased p-AMPK activation upon pioglitazone treatment.

Article Snippet: The blots were blocked in 5% non-fat dried milk and incubated overnight at 4°C with rabbit polyclonal antibodies against ATG5, AMPK, p-AMPK, ULK, p-ULK, mTOR, p-mTOR, PTEN, and p-PTEN (Cell Signaling Technology, Danvers, MA, USA); LC3 (Proteintech, Rosemont, IL); and AAT (Dako, Carpenteria, CA).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Phospho-proteomics, Activation Assay

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Pioglitazone Reduces Hepatic Alpha-1 Antitrypsin Accumulation Through Autophagy and AMPK Activation in Alpha-1 Antitrypsin Deficient Mice

doi: 10.1152/ajpgi.00272.2025

Figure Lengend Snippet: (A) Western blot analysis of liver lysates from control- and pioglitazone-treated Pi*Z mice (30 mg/kg/day, 12 weeks) showing levels of total AAT, ATG5, LC3B-I/II, and p62. GAPDH is shown as a loading control. (B) Quantification of Western blot band intensities from (A), normalized to GAPDH. Data represents SD from 4 mice per group. Statistical significance assessed by unpaired t-test. (C) Immunohistochemistry for LC3B in liver sections from control and pioglitazone-treated mice. The right panel shows higher magnification with arrows indicating LC3B-positive vacuoles. (D) Immunohistochemistry for p62 in liver sections from control and pioglitazone-treated mice. p62-positive aggregates are reduced in pioglitazone-treated livers. Scale bars: 100 μm (all panels).

Article Snippet: The blots were blocked in 5% non-fat dried milk and incubated overnight at 4°C with rabbit polyclonal antibodies against ATG5, AMPK, p-AMPK, ULK, p-ULK, mTOR, p-mTOR, PTEN, and p-PTEN (Cell Signaling Technology, Danvers, MA, USA); LC3 (Proteintech, Rosemont, IL); and AAT (Dako, Carpenteria, CA).

Techniques: Western Blot, Control, Immunohistochemistry

Journal: International dental journal

Article Title: Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux.

doi: 10.1016/j.identj.2023.09.005

Figure Lengend Snippet: Fig. 3 – Role of autophagy in the Yunnan Baiyao (YNBY)−mediated inhibition of osteoclast differentiation. A, B, GFP-RFP-LC3 fluorescence staining revealed that RANKL increased the formation of autophagolysosomes, whilst excluding the effect of solvent dimethylsulfoxide on autophagic flux. C−F, YNBY prevented osteoclast differentiation and F-actin ring formation by inhibiting autophagy. *P < .05. **P < .01.

Article Snippet: Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).

Techniques: Inhibition, Staining, Solvent

Journal: International dental journal

Article Title: Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux.

doi: 10.1016/j.identj.2023.09.005

Figure Lengend Snippet: Fig. 4 – Effect of Yunnan Baiyao (YNBY) on the autophagic flux during osteoclast differentiation. A, B, Bone marrow−derived macrophage (BMM) cells were treated with 20 mg/mL YNBY, 1 mmol/L 3-methyladenine (3-MA), or 100 nmol/L rapamycin (RAP) for 6 hours, and then cells were stained with LysoTracker Red and monitored using confocal immunofluorescence microscopy (scale bar = 100 mm). C, Transmission electron microscopy images of BMMs; left panels (magnification, 10,000x, scale bar = 2 mm) and right panels (magnification, 25,000£, scale bar = 500 nm). Autophagosomes are indicated by yellow arrows. D, E, After infecting BMMs with GFP-RFP-LC3 tandem fluorescent protein adenovirus, the cells were incubated with osteoclast medium supplemented with 20 mg/mL YNBY, 1 mmol/L 3-MA, or 100 nmol/L RAP for 6 hours. Then, fluorescence was observed with a confocal microscope, and quantitative analysis was performed. Scale bar = 100 mm. The data are pre- sented as mean § SDs of 3 independent experiments (*P < .05, **P < .01). All the experiments were carried out independently at least 3 times (*P < .05, **P < .01; bar = 100 mm).

Article Snippet: Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).

Techniques: Derivative Assay, Staining, Microscopy, Transmission Assay, Electron Microscopy, Incubation

Journal: International dental journal

Article Title: Yunnan Baiyao Inhibits Periodontitis by Suppressing the Autophagic Flux.

doi: 10.1016/j.identj.2023.09.005

Figure Lengend Snippet: Fig. 5 – Yunnan Baiyao (YNBY) inhibited bone resorption and the expression of osteoclast-related proteins and autophagy- related proteins. A, B, Bone marrow−derived macrophages (BMMs) were seeded on bovine bone slices and allowed to adhere to the surface. BMMs were pretreated with 1 mmol/L 3-methyladenine (3-MA) or 100 nmol/L rapamycin (RAP) for 1 hour and then cultured with osteoclast medium for 5 days. Representative scanning electron microscopy images of bone resorption pits are shown. C, D, BMMs were pretreated with 1 mmol/L 3-MA or 100 nmol/L RAP for 1 hour and then cultured with RANKL for 6 hours. The total proteins were extracted to measure the levels of P62 and LC3. The total proteins were extracted on the 5th day, and western blotting was performed to examine the expression of the osteoclast-related proteins NFATc-1 and CTSK. E, Schematic model of the hypothesised mechanism by which YNBY inhibits osteoclast differentiation. Densitometric analysis of an immunoblot from 3 independent experiments. *P < .05. **P < .01.

Article Snippet: Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).

Techniques: Expressing, Derivative Assay, Cell Culture, Electron Microscopy, Western Blot

Journal: Nutrients

Article Title: Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.

doi: 10.3390/nu13093026

Figure Lengend Snippet: Figure 1. The phenotype at birth and early postnatal lethality with starvation of liver-specific Atg5-deficient and Control newborn mice. (A) The birth weight of KO (Atg5flox/flox; Albumin-Cre+) and Control (Atg5flox/+; Albumin-Cre+) mice. The birth weight (mean ± SEM) was not significantly different between the groups. The number of mice were 50 (KO) and 45 (Control). (B) Photograph of a representative KO mouse compared with a Control littermate. No abnormal appearance was noted in the KO or Control newborns. (C) Kaplan-Meier curves of KO and Control mice. There was no significant difference in the survival rates up to 22 h of starvation. KO: liver-specific Atg5-deficient; n.s.: not significant.

Article Snippet: As the primary antibodies, we used rabbit polyclonal antibodies against ATG5 (NB110-53818; NOVUS; 1:1000, pretreated by heating), LC3B (ab51520; abcam; 1:10,000, pre-treated by heating), and guinea pig polyclonal antibody against p62 (C-terminal) (GP62-C; PROGEN; 1:500, pre-treated by heating).

Techniques: Control

Journal: Nutrients

Article Title: Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.

doi: 10.3390/nu13093026

Figure Lengend Snippet: Figure 2. The Western blotting and immunohistochemical analyses of ATG5, LC3, and p62 in liver extract. (A) The protein expression of the ATG5, LC3 and p62 in liver extract after 1, 3, 6, 9, and 12 h starvation. β-actin was proceeded in parallel gel and used as an internal control. (B,C) The relative LC3-II/LC3-I ratio (B) and p62 level (C) are shown in graphs. The numbers of mice (starvation time) were as follows: 5 (1 h), 5 (3 h), 5 (6 h), 5 (9 h), and 6 (12 h) Control mice and 4 (1 h), 5 (3 h), 5 (6 h), 5 (9 h), and 5 (12 h) KO mice. Each error bar is expressed as the mean ± SEM. Student’s t-test; * p < 0.05, ** p < 0.01. (D) ATG5, LC3B and p62 immunostainings of liver samples of KO mice and Control mice after 12 h starvation. (E) Liver cells with intracytoplasmic dot-like structures labelled by anti-LC3B antibody. The LC3B expression was decreased in KO mice (F) Dots labelled by anti-p62 antibody. The expression of p62 were significantly increased during starvation in KO mice. Samples were immunostained and then counterstained with hematoxylin. Bar = 100 µm for ATG5, 10 µm for LC3B, and 20 µm for p62. KO: liver-specific Atg5-deficient; ATG5: autophagy related 5; LC3: microtubule-associated protein 1A/1B-light chain 3; LC3-I; cytosolic form of LC3; LC3-II: phosphatidylethanolamine conjugate form of LC3; LC3B: LC3 microtubule-associated protein 1A/1B-light chain 3B; HPF: high power fields.

Article Snippet: As the primary antibodies, we used rabbit polyclonal antibodies against ATG5 (NB110-53818; NOVUS; 1:1000, pretreated by heating), LC3B (ab51520; abcam; 1:10,000, pre-treated by heating), and guinea pig polyclonal antibody against p62 (C-terminal) (GP62-C; PROGEN; 1:500, pre-treated by heating).

Techniques: Western Blot, Immunohistochemical staining, Expressing, Control

Journal: Nutrients

Article Title: Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.

doi: 10.3390/nu13093026

Figure Lengend Snippet: Figure 3. The gas chromatography-tandem mass spectrometry analysis of metabolites in the serum of starved neonatal mice. The numbers of mice (starvation time) were as follows: 5 (1 h), 6 (3 h), 5 (6 h), 6 (9 h), and 6 (12 h) Control mice and 4 (1 h), 5 (3 h), 7 (6 h), 7 (9 h), and 5 (12 h) KO mice. The metabolite concentrations were measured as the peak area of the calibration curve, and the concentration at each starvation time was compared between the groups. The levels of serum saccharides and glycerol (A), ketone bodies (B), and representative metabolites of the TCA cycle (C) during starvation. Boxes represent the interquartile range (25th to 75th percentiles), and lines within the boxes are the median; error bars represent the 25th percentile minus 1.5 times the interquartile range (IQR) and the 75th percentile plus 1.5 times the IQR. Mann-Whitney U test; * p < 0.05, KO: liver-specific Atg5-deficient.

Article Snippet: As the primary antibodies, we used rabbit polyclonal antibodies against ATG5 (NB110-53818; NOVUS; 1:1000, pretreated by heating), LC3B (ab51520; abcam; 1:10,000, pre-treated by heating), and guinea pig polyclonal antibody against p62 (C-terminal) (GP62-C; PROGEN; 1:500, pre-treated by heating).

Techniques: Gas Chromatography, Mass Spectrometry, Control, Concentration Assay, MANN-WHITNEY

Journal: Nutrients

Article Title: Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.

doi: 10.3390/nu13093026

Figure Lengend Snippet: Figure 5. Multivariate statistical and pathway analyses of serum metabolites after 12 h of starvation. (A) The OPLS-DA score scatter plot generated from serum metabolite data of KO and Control mice. The x-axis shows the intra-group variance, and the y-axis shows the inter-group variance. Each point represents the results of an analysis of data from one mouse. A differential metabolic pattern was noted between KO (blue) and Control group (green) by the plots clearly separated on the score scatter plot. (B) S-plot of serum metabolites from KO and Control mice. Each dot indicates individual metabolite. The numbers beside the dots indicate the individual numbers of metabolites. The S-plot visualizes both the covariance (x-axis) and correlation (y-axis, p-value) between the metabolites and the modelled class designation. The 52 significantly altered metabolites with 0.7 < p (corr) <1.0 (red dots) or –1.0 < p (corr) < –0.7 (blue dot) were selected as potential biomarker metabolites (shown in Supplementary Table S2.). (C) A MetPA based on potential biomarker metabolites. The MetPA visualizes the pathway impact (x-axis), p-value (y-axis, colour of the circle), and number of hit metabolites (width of circle). (D) A MSEA based on potential biomarker metabolites. Color intensity (yellow to red) reflects increasing statistical significance MSEA. The lengths of the bars represent the fold enrichment. Six pathways were identified as significantly changed metabolic pathways affected by liver-specific autophagy-deficiency (Ammonia Recycling, Malate-Aspartate Shuttle, Homocysteine Degradation, Aspartate Metabolism, Urea Cycle and Glycine and Serine Metabolism). KO: liver-specific Atg5-deficient; OPLS-DA: orthogonal partial least squares discriminant analysis; MetPA: metabolic pathway analysis; MSEA: metabolite set enrichment analysis.

Article Snippet: As the primary antibodies, we used rabbit polyclonal antibodies against ATG5 (NB110-53818; NOVUS; 1:1000, pretreated by heating), LC3B (ab51520; abcam; 1:10,000, pre-treated by heating), and guinea pig polyclonal antibody against p62 (C-terminal) (GP62-C; PROGEN; 1:500, pre-treated by heating).

Techniques: Generated, Control, Biomarker Discovery

Journal: Nutrients

Article Title: Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.

doi: 10.3390/nu13093026

Figure Lengend Snippet: Figure 6. Ultrastructural demonstration of mitochondria in hepatocytes of Control and KO mice after 1 and 12 h of starvation. (A) Representative structural findings of mitochondria. After 1 h of starvation, the mitochondrial structures were almost normal in both KO and control mice. Damaged mitochondria, such as those with dysmorphic cristae and abnormal inclusion, were noticed in KO mice after 12 h of starvation. Bar = 1 µm. N: nucleus. (B–E) Quantification of mitochondrial length (B), width (C), aspect ratio (D) and area (E) in hepatic cells of Control and KO group at 1 h and 12 h starvation (mean ± SEM). Number of mice/cells/mitochondria of Control and KO group at 1 h starvation were 3/6/147 and 3/6/172, respectively. Number of mice/cells/mitochondria of Control and KO group at 12 h starvation were 3/6/157 and 3/6/136, respectively. Student T test; * p < 0.05, ** p < 0.01, *** p < 0.001. KO: liver-specific Atg5-deficient.

Article Snippet: As the primary antibodies, we used rabbit polyclonal antibodies against ATG5 (NB110-53818; NOVUS; 1:1000, pretreated by heating), LC3B (ab51520; abcam; 1:10,000, pre-treated by heating), and guinea pig polyclonal antibody against p62 (C-terminal) (GP62-C; PROGEN; 1:500, pre-treated by heating).

Techniques: Control

Journal: Cells

Article Title: High Dietary Phosphate Exacerbates and Acts Independently of Low Autophagy Activity in Pathological Cardiac Remodeling and Dysfunction

doi: 10.3390/cells10040777

Figure Lengend Snippet: Absence of atg5 in cardiomyocytes induced short lifespan and cardiac dysfunction. ( A ) The changes in autophagic markers and atg5 in the hearts of H-atg5 −/− mice and WT mice at 3 months of age. Left panel: Representative immunoblots for atg5, LC3, and p62 in left ventricular lysates. Right panel: Summary of all immunoblot data. Quantitative data are presented as mean ± SD with scatter plots of individual data points, and statistical significance was assessed by unpaired Student’s t -test. Significant differences are accepted when ** p < 0.01 between two groups. ( B ) Kaplan–Meier survival curves for H-atg5 −/− and WT mice. ( C ) Left ventricular ejection fraction determined by cardiac MRI in H-atg5 −/− mice and WT mice at 3 and 6 months of age. Left ventricular wall thickness at systole ( D ) and at diastole ( E ) determined by cardiac MRI in H-atg5 −/− mice and WT mice at 3 and 6 months old. Sample number in each group is presented in brackets underneath corresponding bar. Quantitative data are presented as mean ± SD with scatter plots of individual data points, and statistical significance was assessed by two-way ANOVA followed by Newman–Keuls test. * p < 0.05, ** p < 0.01 vs. WT mice at the same age; # p < 0.05 vs. WT at 3 months old; $ p < 0.05, $$ p < 0.01 vs. H-atg5 −/− mice at 3 months old.

Article Snippet: The following antibodies were used for immunoblotting and/or immunohistochemistry: mouse monoclonal antibody against α-actin (MAB1501, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal antibody against α-actinin (A7732, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody against atg5 (NB110-53818, Novus, Centennial, CO, USA); rabbit monoclonal antibody against cleaved caspase-3 (#9661, CST, Danvers, MA, USA); rabbit polyclonal antibody against LC3 (NB100-2220, Novus, Centennial, CO, USA); mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan); mouse monoclonal antibody against α-SMA (A5528, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot

Journal: Cells

Article Title: High Dietary Phosphate Exacerbates and Acts Independently of Low Autophagy Activity in Pathological Cardiac Remodeling and Dysfunction

doi: 10.3390/cells10040777

Figure Lengend Snippet: Cardiac hypertrophy and fibrosis in H-atg5 −/− mice. ( A ) Representative images of the hearts of H-atg5 −/− mice and WT mice at 3 and 6 months old. ( B ) Heart weight/body weight in H-atg5 −/− mice and WT mice at 3 and 6 months of age. ( C ) Representative macrographs (upper panel) and micrographs (middle panel) of sagittal TC-stained sections of the hearts of H-atg5 −/− and WT mice at 3 and 6 months of age. Semi-quantification (bottom panel) of the TC-positive area over the whole-heart section with Image J software. ( D ) Changes in hypertrophic and fibrotic markers in the hearts of H-atg5 −/− mice and WT mice at the age of 3 months. Left panel: representative immunoblots for α-actinin and α-SMA in left ventricular lysates. Right panel: a summary of all immunoblot data. ( E ) Quantitative analysis of transcripts of β-MHC, α-SMA, and α-actinin in left ventricular lysates in H-atg5 −/− mice and WT mice at the age of 3 months. Sample number in each group is presented in brackets underneath corresponding bars. Quantitative data are presented as mean ± SD with scatter plots of individual data points. * p < 0.05, ** p < 0.01 vs. WT mice at the same age; # p < 0.05 vs. WT at 3 months old; $ p < 0.05, $$ p < 0.01 vs. H-atg5 −/− mice at 3 months old by two-way ANOVA followed by Student–Newman–Keuls post-hoc test.

Article Snippet: The following antibodies were used for immunoblotting and/or immunohistochemistry: mouse monoclonal antibody against α-actin (MAB1501, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal antibody against α-actinin (A7732, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody against atg5 (NB110-53818, Novus, Centennial, CO, USA); rabbit monoclonal antibody against cleaved caspase-3 (#9661, CST, Danvers, MA, USA); rabbit polyclonal antibody against LC3 (NB100-2220, Novus, Centennial, CO, USA); mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan); mouse monoclonal antibody against α-SMA (A5528, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Staining, Software, Western Blot

Journal: Cells

Article Title: High Dietary Phosphate Exacerbates and Acts Independently of Low Autophagy Activity in Pathological Cardiac Remodeling and Dysfunction

doi: 10.3390/cells10040777

Figure Lengend Snippet: High dietary phosphate increased mortality in H-atg5 −/− mice. H-atg5 −/− mice were fed with normal- or high-phosphate diet starting at 12 weeks old for 12 weeks. ( A ) Plasma phosphate (Pi) concentration after 12-week dietary phosphate treatment in H-atg5 −/− mice; ( B ) Fractional excretion of phosphate after 12-week dietary phosphate treatment; ( C ) Kaplan–Meier survival curves of H-atg5 −/− mice fed with normal- or high-phosphate diet; ( D ) Heart weight/body weight in H-atg5 −/− mice fed with normal- or high-phosphate diet for 12 weeks. Sample number in each group is presented in brackets underneath corresponding bar. Quantitative data are presented as mean ± SD with scatter plots of individual data points, and statistical significance was assessed by unpaired Student’s t -test. Significant differences were accepted when * p < 0.05 or ** p < 0.01 between groups. FE Pi : fractional excretion of phosphate; H Pi: high-phosphate diet; N Pi: normal-phosphate diet.

Article Snippet: The following antibodies were used for immunoblotting and/or immunohistochemistry: mouse monoclonal antibody against α-actin (MAB1501, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal antibody against α-actinin (A7732, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody against atg5 (NB110-53818, Novus, Centennial, CO, USA); rabbit monoclonal antibody against cleaved caspase-3 (#9661, CST, Danvers, MA, USA); rabbit polyclonal antibody against LC3 (NB100-2220, Novus, Centennial, CO, USA); mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan); mouse monoclonal antibody against α-SMA (A5528, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Clinical Proteomics, Concentration Assay

Journal: Cells

Article Title: High Dietary Phosphate Exacerbates and Acts Independently of Low Autophagy Activity in Pathological Cardiac Remodeling and Dysfunction

doi: 10.3390/cells10040777

Figure Lengend Snippet: High phosphate exacerbated pathological cardiac remodeling in H-atg5 −/− mice. WT mice or H-atg5 −/− mice were fed with normal- or high-phosphate diet starting at 3 months old for 12 weeks. ( A ) Representative macrographs (upper panel) and micrographs (bottom panel) of sagittal TC-stained sections of the hearts. ( B ) Semi-quantification of the TC-positive area over the whole-heart section with Image J. ( C ) Changes in hypertrophic and fibrotic markers in the heart. Left panel: representative immunoblots of left ventricular lysates for α-actinin and α-SMA protein. Right panel: a summary of all immunoblot data. ( D ) Hypertrophic cardiomyocytes in free walls of left ventricles of mice. Upper panel: representative immunohistochemistry for WGA. Bottom panel: semi-quantification of myocyte size with Image J. ( E ) The changes in apoptotic markers in the heart. Upper panel: representative immunoblots for cleaved caspase-3 in left ventricular lysates. Bottom panel: a summary of immunoblot data. ( F ) Changes in apoptotic markers in the heart. Upper panel: representative immunofluorescent images for TUNEL in free wall of left ventricles. Bottom panel: semi-quantification of TUNEL-positive cells/DAPI-positive cardiomyocytes. Sample number in each group is presented in brackets underneath corresponding bars. Quantitative data are presented as mean ± SD with scatter plots of individual data points. * p < 0.05, ** p < 0.01 vs. WT mice at the same dietary phosphate treatment; # p < 0.05, ## p < 0.01 vs. WT treated with normal-phosphate diet; $ p < 0.05, $$ p < 0.01 vs. H-atg5 −/− mice treated with normal-phosphate diet. Statistical significance was assessed by two-way ANOVA followed by Newman–Keuls test. H Pi: high-phosphate diet; N Pi: normal-phosphate diet.

Article Snippet: The following antibodies were used for immunoblotting and/or immunohistochemistry: mouse monoclonal antibody against α-actin (MAB1501, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal antibody against α-actinin (A7732, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody against atg5 (NB110-53818, Novus, Centennial, CO, USA); rabbit monoclonal antibody against cleaved caspase-3 (#9661, CST, Danvers, MA, USA); rabbit polyclonal antibody against LC3 (NB100-2220, Novus, Centennial, CO, USA); mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan); mouse monoclonal antibody against α-SMA (A5528, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Staining, Western Blot, Immunohistochemistry, TUNEL Assay

Journal: Cells

Article Title: High Dietary Phosphate Exacerbates and Acts Independently of Low Autophagy Activity in Pathological Cardiac Remodeling and Dysfunction

doi: 10.3390/cells10040777

Figure Lengend Snippet: High phosphate induced similar severity of kidney fibrosis between WT and H-atg5 −/− mice. WT and H-atg5 −/− mice were fed with normal- or high-phosphate diet starting at 3 months old for 12 weeks. ( A ) Plasma creatinine (Cr). ( B ) Kidney fibrosis was determined in TC-stained sections. Left panel: Representative micrographs of TC-stained kidney sections. Black arrows depict tubulointerstitial fibrosis. Right panel: Semi-quantification of the TC-positive area over the kidney section with Image J. Sample number in each group is presented in brackets underneath corresponding bar. Quantitative data are presented as mean ± SD with scatter plots of individual data points. ## p < 0.01 vs. WT treated with normal-phosphate diet; $$ p < 0.01 vs. H-atg5 −/− mice treated with normal-phosphate diet. Statistical significance was assessed by two-way ANOVA followed by Newman–Keuls test. H Pi: high-phosphate diet; N Pi: normal-phosphate diet.

Article Snippet: The following antibodies were used for immunoblotting and/or immunohistochemistry: mouse monoclonal antibody against α-actin (MAB1501, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal antibody against α-actinin (A7732, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody against atg5 (NB110-53818, Novus, Centennial, CO, USA); rabbit monoclonal antibody against cleaved caspase-3 (#9661, CST, Danvers, MA, USA); rabbit polyclonal antibody against LC3 (NB100-2220, Novus, Centennial, CO, USA); mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan); mouse monoclonal antibody against α-SMA (A5528, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Clinical Proteomics, Staining

Journal: Cells

Article Title: High Dietary Phosphate Exacerbates and Acts Independently of Low Autophagy Activity in Pathological Cardiac Remodeling and Dysfunction

doi: 10.3390/cells10040777

Figure Lengend Snippet: High phosphate exacerbates atg5 deficiency-induced cardiac remodeling. High phosphate decreases autophagy activity in cardiomyocytes. Atg5 deletion in cardiomyocytes downregulates autophagy activity in cardiomyocytes. Low autophagy activity results in cardiomyocyte apoptosis. High phosphate also induces cardiomyocyte apoptosis, which is autophagy-independent. Increase in apoptosis in cardiomyocytes leads to cardiomyocyte damage and cardiac hypertrophy and fibrosis. Therefore, high phosphate and low autophagy individually trigger and additionally promote heart deterioration. However, high-phosphate-induced pathologic cardiac remodeling might be autophagy-independent and apoptosis-independent (dash line), whose mechanisms are not explored in the current study. On the other hand, low autophagy can induce cardiac remodeling independently of apoptosis (dash line), which is worth illustrating.

Article Snippet: The following antibodies were used for immunoblotting and/or immunohistochemistry: mouse monoclonal antibody against α-actin (MAB1501, Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal antibody against α-actinin (A7732, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody against atg5 (NB110-53818, Novus, Centennial, CO, USA); rabbit monoclonal antibody against cleaved caspase-3 (#9661, CST, Danvers, MA, USA); rabbit polyclonal antibody against LC3 (NB100-2220, Novus, Centennial, CO, USA); mouse monoclonal antibody against p62 (H00008878-M01, Abnova, Taipei, Taiwan); mouse monoclonal antibody against α-SMA (A5528, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activity Assay