Journal: Frontiers in pharmacology

Article Title: Activating Cannabinoid Receptor 2 Protects Against Diabetic Cardiomyopathy Through Autophagy Induction.

doi: 10.3389/fphar.2018.01292

Figure Lengend Snippet: FIGURE 2 | Administration of HU308 enhances cardiac autophagy in heart issues from DCM mice. (A) Representative images of the autophagy-related proteins including beclin-1, LC3-II/I, and p62. (B) Quantitative analysis of the autophagy-related proteins (n = 5). ∗∗P < 0.01 vs. Nor group; $$P < 0.01 vs. DCM group. Nor, normal; DCM, diabetic cardiomyopathy.

Article Snippet: The primary antibodies applied were listed as follows: rabbit anti-Beclin-1 monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-LC3 polyclonal antibody (1:1000; Novus Biologicals, Littleton, CO, United States), rabbit anti-p62 antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-adenosine 5′- monophosphate (AMP)-activated protein kinase (AMPK) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated AMPK antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) anti-mammalian target of rapamycin rabbit (mTOR) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated mTOR antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-p70 ribosomal protein S6 kinase (p70S6K) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit antiphosphorylated p70S6K antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) and mouse anti-β-actin antibody (1:5000, Beyotime Biotechnology, Shanghai, China).

Techniques:

Journal: Frontiers in pharmacology

Article Title: Activating Cannabinoid Receptor 2 Protects Against Diabetic Cardiomyopathy Through Autophagy Induction.

doi: 10.3389/fphar.2018.01292

Figure Lengend Snippet: FIGURE 4 | Administration of HU308 enhances cardiac autophagy in cardiomyocytes under the challenge of HG. Primary murine ventricular cardiomyocytes were obtained from neonatal C57BL/6J mice and challenged with high glucose concentration (HG; 33 mM glucose) for 24 h as hyperglycemia group or medium with normal glucose concentration (Nor; 5.5 mM glucose) as the control group. In certain groups, HU308 (10 µM) was treated to cells at 10 min before suffering HG. PBS in the same volume was administrated as vehicle. (A) Representative images of the autophagy-related proteins including beclin-1, LC3-II/I, and p62. (B) Quantitative analysis of the autophagy-related proteins (n = 5). ∗∗P < 0.01 vs. Nor group; $$P < 0.01 vs. HG group. (C) Representative images of transmission electron microscopy with arrows marking autophagosomes. (D) Quantitative analysis of the number of autophagosomes in murine ventricular cardiomyocytes (n = 5). ∗∗P < 0.01 vs. Nor group; $$P < 0.01 vs. HG group. Nor, normal; DCM, diabetic cardiomyopathy.

Article Snippet: The primary antibodies applied were listed as follows: rabbit anti-Beclin-1 monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-LC3 polyclonal antibody (1:1000; Novus Biologicals, Littleton, CO, United States), rabbit anti-p62 antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-adenosine 5′- monophosphate (AMP)-activated protein kinase (AMPK) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated AMPK antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) anti-mammalian target of rapamycin rabbit (mTOR) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated mTOR antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-p70 ribosomal protein S6 kinase (p70S6K) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit antiphosphorylated p70S6K antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) and mouse anti-β-actin antibody (1:5000, Beyotime Biotechnology, Shanghai, China).

Techniques: Concentration Assay, Control, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Endoplasmic reticulum tubules limit the size of misfolded protein condensates

doi: 10.7554/eLife.71642

Figure Lengend Snippet: List of antibodies used in this study.

Article Snippet: Rabbit anti-LC3 pAb , MBL , PM036 , 1:1000.

Techniques: Control, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 2-Hydroxypropyl-β-cyclodextrin Promotes Transcription Factor EB-mediated Activation of Autophagy

doi: 10.1074/jbc.M113.506246

Figure Lengend Snippet: HPβCD treatment results in activation of autophagy. A, confocal microscopy analysis of LC3 expression in HeLa/TFEB cells transfected for the expression of LC3-GFP for 20 h and treated with 1 mm HPβCD for additional 24 h. The scale bar is 10 μm. UT, untreated. B, Western blot analyses of LC3 isoforms and GAPDH (used as loading control) in HeLa/TFEB cells treated with 1 mm HPβCD and 100 nm bafilomycin for 24 h and quantification of LC3-II bands. Band intensities were quantified with ImageJ analysis software, corrected by GAPDH band intensities, and divided by the values obtained in untreated samples (p < 0.01; *, p < 0.05). Baf, bafilomycin. C, Western blot analyses of LC3 isoforms and GAPDH (used as loading control) in HeLa/TFEB cells treated with 1 mm HPβCD for 0, 12, 24, 48, and 72 h and quantification of LC3-II/LC3-I ratios at each time point. Band intensities were quantified as described in A (*, p < 0.05). D, relative mRNA expression levels of representative genes of the autophagy system in HeLa cells stably transfected with TFEB-FLAG treated with 1 mm HPβCD for days. mRNA expression levels of MAPLC3B, SQSTM1, and BECN1 were obtained as described in Fig. 1. Data are reported as the mean ± S.D. n ≥ 3; p < 0.01.

Article Snippet: Cells were permeabilized with 0.1% Triton X for 5 min and incubated with 8% BSA for 1 h. After incubation for 1 h with primary antibodies (rabbit anti-3×FLAG (Sigma), rabbit anti-LC3 (MBL International), mouse anti-LAMP2 (BioLegend), or mouse anti-TFEB (Abcam)), cells were washed 3 times with 0.1% Tween 20, PBS and incubated with secondary antibodies for 1 h (Dylight 549 goat anti-mouse IgG or Dylight 633 goat anti-rabbit IgG (KPL)).

Techniques: Activation Assay, Confocal Microscopy, Expressing, Transfection, Western Blot, Software, Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: 2-Hydroxypropyl-β-cyclodextrin Promotes Transcription Factor EB-mediated Activation of Autophagy

doi: 10.1074/jbc.M113.506246

Figure Lengend Snippet: HPβCD treatment activates autophagic clearance. A, confocal microscopy analysis of ceroid lipopigment (green) and LC3 (red) in LINCL fibroblasts treated with 1 mm HPβCD and 100 nm bafilomycin for 3 days, evaluated by detecting green autofluorescence and binding of an anti-LC3 antibody, respectively. The scale bar is 20 μm. UT, untreated; Baf, bafilomycin. B, Western blot analyses of LC3 isoforms and GAPDH (used as loading control) in LINCL fibroblasts treated with 1 mm HPβCD and 100 nm bafilomycin for 24 h and quantification of LC3-II bands. Band intensities were quantified with ImageJ analysis software, corrected by GAPDH band intensities, and divided by the values obtained in untreated samples (p < 0.05). C, confocal microscopy analysis of ceroid lipopigment (green, first column), LC3 (red, second column), and LAMP-2 (blue, third column) in LINCL fibroblasts treated with 1 mm HPβCD and 100 nm bafilomycin for 3 days, evaluated by detecting green autofluorescence, binding of anti-LC3 antibody, and binding of anti-LAMP-2 antibody, respectively. Co-localization of LC3 and LAMP-2 is shown in purple (fourth column). Heatmaps of co-localization images were obtained with ImageJ analysis software (fifth column). Hot colors represent positive correlation (co-localization), whereas cold colors represent negative correlation (exclusion). The scale bar is 20 μm.

Article Snippet: Cells were permeabilized with 0.1% Triton X for 5 min and incubated with 8% BSA for 1 h. After incubation for 1 h with primary antibodies (rabbit anti-3×FLAG (Sigma), rabbit anti-LC3 (MBL International), mouse anti-LAMP2 (BioLegend), or mouse anti-TFEB (Abcam)), cells were washed 3 times with 0.1% Tween 20, PBS and incubated with secondary antibodies for 1 h (Dylight 549 goat anti-mouse IgG or Dylight 633 goat anti-rabbit IgG (KPL)).

Techniques: Confocal Microscopy, Binding Assay, Western Blot, Software

Journal: Journal of Ovarian Research

Article Title: Human recombinant arginase I [HuArgI (Co)-PEG5000]-induced arginine depletion inhibits ovarian cancer cell adhesion and migration through autophagy-mediated inhibition of RhoA

doi: 10.1186/s13048-021-00767-3

Figure Lengend Snippet: Arginine depletion-mediated decrease in cell adhesion and motility is through the stimulation of autophagy in ovarian cancer cells. a Left panel: Micrographs illustrating SKOV3 cells treated with hArgI or rapamycin and stained with Cyto-ID (green). Fluorescent images were taken using the 63x objective lens of the Zeiss Observer Z1 fluorescent microscope. Scale bar is 10 μm. b LC3 cleavage in SKOV3 (upper panel) and Caov-3 (lower panel) and actin blot for loading control (lower gels) following treatment with hArgI or rapamycin on western blot. c SKOV3 cells were treated with hArgI, rapamycin or hArgI in combination with chloroquine before being imaged moving randomly in media. Cell images were captured at 1 min interval for 2 h.The average migrated distance (in μm) was measured by tracking the images using ImageJ software and the speed was calculated by dividing the distance over time. The bar graph illustrates the fold change in migration of hArgI, rapamycin or hArgI + chloroquine treated SKOV3 cells normalized to the untreated control. d Representative micrographs of fixed and stained control SKOV3 cells, and SKOV3 cells treated with either hArgI, rapamycin or hArgI in combination with chloroquine. Scale bar is 100 μm. Right panel: Colorimetric quantification of the dissolved crystal violet stain of the cells described in C was performed at 560 nm using an Elisa plate reader. The bar graph represents the quantification of adherent cells expressed as fold increase relative to the untreated control. Significance was set at p < 0.05. e Bar graph illustrating the number of focal adhesions in control and hArgI or rapamycin treated cells stained with anti-vinculin. The number of focal adhesion was quantified using the CLAHE and Log3D plugins in Image and is expressed as absolute values of the means for every sample

Article Snippet: Rabbit polyclonal anti-LC3 antibody was obtained from Cell Signaling (Cell Signaling Technology Inc., US).

Techniques: Staining, Microscopy, Control, Western Blot, Software, Migration, Enzyme-linked Immunosorbent Assay