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: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Observation of microtubule-associated proteins 1A/1B light chain 3 (LC3) and actin double-stained fluorescence images of rat embryonic fibroblasts (REF) cells. ( A ) Indirect fluorescein isothiocyanate (FITC) immunofluorescence labelling of LC3 using optical microscope (OM) with a green fluorescence filter (see Methods; 400× magnification). ( B ) Indirect rhodamine immunofluorescence labelling of actin viewed through a red fluorescence filter (see Methods; 400× magnification). An enlarged image of the white framed area is shown in the upper corner. ( C ) A merged fluorescence image of LC3 and actin. ( D , E ) Enlarged images of the LC3-rich areas (indicated by yellow arrows in C ). ( F , G ) Enlarged images of the LC3-poor areas (indicated by white arrows in ( C )) showing many filamentous actin bundles. ( H ) A merged image of LC3 and phalloidin-labelled filamentous actin. ( I , J ) Enlarged images of the LC3-rich areas (indicated by yellow arrows in ( H )). ( K , L ) Enlarged images of the LC3-poor areas (indicated by white arrows in ( H )). Scale bars: 50 μm in ( A – C , H ), 10 μm in ( D–G , I–L ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining, Fluorescence, Immunofluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Observation of Atg12 and actin double-stained fluorescence images of REF cells. ( A ) Indirect FITC immunofluorescence labelling of Atg12 using OM with a green fluorescence filter (see Materials and Methods; 400× magnification). Small fluorescence spots near the nucleus are Atg12. ( B ) A fluorescence image of actin filament stained by rhodamine-conjugated phalloidin, viewed through a red fluorescence filter (400× magnification). ( C ) A merged fluorescence image of Atg12 and actin. ( D , E ) Enlarged images of the Atg12-rich areas (indicated by yellow arrows in ( C )). ( F , G ) Enlarged images of the Atg12-poor areas (indicated by white arrows in ( C )). Fluorescence spots of Atg12 are rare near the filamentous actin. ( H , I ) Enlarged images of the LC3 and Atg12 fluorescence spots, respectively. Scale bars: 50 μm in ( A – C ), 10 μm in ( D – I ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining, Fluorescence, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Dielectric images of 4T1E/M3 cells stained with anti-LC3 antibody conjugated to 60 nm colloidal gold particles. ( A ) Dielectric images of the cells stained with anti-LC3 antibody conjugated to 60 nm colloidal gold particles after paraformaldehyde fixation and permeabilization (10,000× magnification, 6 kV electron beam (EB)). ( B ) An enlarged image of the red framed area in ( A ), showing LC3 bound to 60 nm colloidal gold particles. ( C ) A pseudo-color map of ( B ) after intensity inversion. ( D ) A schematic diagram of arc-like structures and colloidal gold particles superimposed on a dielectric image of ( B ). ( E ) A 3D color map of ( C ). ( F ) Another scanning electron-assisted dielectric microscopy (SE-ADM) image of 4T1E/M3 cells (10,000× magnification, 6 kV EB). ( G ) An enlarged image of the red framed area in ( E ). ( H ) A pseudo-color map of ( F ) after intensity inversion. LC3 aligns in a circular manner. ( I ) A 3D color map of ( H ). Scale bars: 1 μm in ( A , F ), 500 nm in ( B – E , G – I ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Dielectric images of REF cells stained with anti-Atg12 antibody conjugated to 60 nm colloidal gold particles. ( A ) A low-magnification dielectric image of the cells stained with anti-Atg12 antibody conjugated to 60 nm colloidal gold particles after fixation and permeabilization (1500× magnification, 6 kV EB). ( B ) A high-magnification image (10,000×) of the red framed area in ( A ), showing Atg12 conjugated to 60 nm colloidal gold particles. ( C ) An enlarged image of the red framed area in ( B ). ( D ) A pseudo-color map of ( C ) after intensity inversion. ( E ) A schematic diagram of meshwork structures and colloidal gold particles superimposed on a dielectric image of ( C ). ( F ) A 3D color map of ( D ). ( G ) Histograms of minimum intervals between pairs of colloidal gold particles. Pairs of particles (183 for LS3 and 300 for Atg12) were randomly selected and the intervals were measured. Minimum intervals between LC3-bound colloids are small and sharply peaked, whereas those between Atg12 are larger and gently distributed. ( H ) A schematic illustration of the structural organization of autophagosomes involving LC3 and Atg12 with actin. Scale bars: 10 μm in ( A ), 1 μm in ( B ), 500 nm in ( C – F ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining

Journal: Neural Regeneration Research

Article Title: Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury

doi: 10.4103/1673-5374.300725

Figure Lengend Snippet: Apelin-13 alleviates autophagy in cerebral ischemia/reperfusion models in vivo and in vitro . (A) Double immunofluorescent staining of LC3B (green, stained by fluorescein isothiocyanate) and NeuN (red, stained by Cy5) in the rat hippocampus. Apelin-13 treatment significantly decreased the expression of LC3B in the hippocampus of MCAO rats. (B) Representative western blots and quantitative evaluations of LC3B in the rat hippocampus. (C) Double immunofluorescent staining of p62 (green, stained by fluorescein isothiocyanate) and NeuN (red, stained by Cy5) in the rat hippocampus. Apelin-13 treatment significantly increased the expression of p62 in the hippocampus of MCAO rats. (D) Representative western blots and quantitative evaluations of p62 in the rat hippocampus. (E) Acridine orange staining of SH-SY5Y cells. Arrows indicate autophagic vacuoles. Scale bars: 500 μm in A and C, 100 μm in E. (F, G) Representative western blots and quantitative evaluations of LC3B and p62 in SH-SY5Y cells. Data are represented as the mean ± standard error of the mean ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . sham or control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . MCAO or OGD/R group (one-way analysis of variance followed by post hoc Tukey’s test). DAPI: 4’,6-Diamidino-2-phenylindole; LC3B: microtubule-associated protein 1 light chain 3; MCAO: middle cerebral artery occlusion; OD: optical density; OGD/R: oxygen-glucose deprivation/reperfusion.

Article Snippet: After blocking at room temperature for 1.5 hours with 5% non-fat milk powder in Tris-buffered saline with Tween-20, the membranes were incubated at 4°C overnight with the following primary antibodies against autophagy and apoptosis markers and other relevant pathway proteins: mouse anti-Bcl-2 (1:1000; Cat# 15071; Cell Signaling Technology), rabbit anti-Bax (1:1000; Cat# 2772; Cell Signaling Technology), rabbit anti-phospho(p)-Akt (1:1000; Cat# 4060; Cell Signaling Technology), mouse anti-Akt (1:1000; Cat# 2920; Cell Signaling Technology), rabbit anti-mTOR (1:1000; Cat# 2983; Cell Signaling Technology), rabbit anti-p-mTOR (1:1000; Cat# ab84400; Cell Signaling Technology), rabbit anti-PI3K (1:1000; Cat# 4292; Cell Signaling Technology), rabbit anti-p-PI3K (1:1000; Bioworld, Nanjing, China), mouse anti-p62 (1:1000; Cat# ab91526; Abcam), rabbit anti-Beclin1 (1:1000; Cat# ab62557; Abcam), rabbit anti-caspase-3 (1:1000; Cat# BS4605; Proteintech, Wuhan, China), rabbit anti-LC3B (1:1000; Cat# NB100-2220; NOVUS), and mouse anti-β-actin (1:2500; Cat# TA-09; Zhongshan Golden Bridge Biotechnology, Beijing China).

Techniques: In Vivo, In Vitro, Staining, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury

doi: 10.4103/1673-5374.300725

Figure Lengend Snippet: LY294002 or rapamycin treatment enhances apoptosis and autophagy in vitro . (A–C) Representative western blots and quantitative evaluations of LC3B (A), p62 (B), and the ratio of Bcl-2/Bax (C) in SH-SY5Y cells. Data are represented as the mean ± standard error of the mean ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . OGD/R group; ++ P < 0.01, +++ P < 0.001, vs . OGD/R + apelin-13 group (one-way analysis of variance followed by post hoc Tukey’s test). LY294002 and rapamycin are inhibitors of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin pathway. LC3B: Microtubule-associated protein 1 light chain 3; OGD/R: oxygen-glucose deprivation/reperfusion.

Article Snippet: After blocking at room temperature for 1.5 hours with 5% non-fat milk powder in Tris-buffered saline with Tween-20, the membranes were incubated at 4°C overnight with the following primary antibodies against autophagy and apoptosis markers and other relevant pathway proteins: mouse anti-Bcl-2 (1:1000; Cat# 15071; Cell Signaling Technology), rabbit anti-Bax (1:1000; Cat# 2772; Cell Signaling Technology), rabbit anti-phospho(p)-Akt (1:1000; Cat# 4060; Cell Signaling Technology), mouse anti-Akt (1:1000; Cat# 2920; Cell Signaling Technology), rabbit anti-mTOR (1:1000; Cat# 2983; Cell Signaling Technology), rabbit anti-p-mTOR (1:1000; Cat# ab84400; Cell Signaling Technology), rabbit anti-PI3K (1:1000; Cat# 4292; Cell Signaling Technology), rabbit anti-p-PI3K (1:1000; Bioworld, Nanjing, China), mouse anti-p62 (1:1000; Cat# ab91526; Abcam), rabbit anti-Beclin1 (1:1000; Cat# ab62557; Abcam), rabbit anti-caspase-3 (1:1000; Cat# BS4605; Proteintech, Wuhan, China), rabbit anti-LC3B (1:1000; Cat# NB100-2220; NOVUS), and mouse anti-β-actin (1:2500; Cat# TA-09; Zhongshan Golden Bridge Biotechnology, Beijing China).

Techniques: In Vitro, Western Blot