Journal: Journal of pharmacological sciences

Article Title: Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

doi: 10.1016/j.jphs.2020.12.001

Figure Lengend Snippet: Fig. 1. (A) Schematic diagram of the experimental design of protocol 1. (B) Representative images of LC3 (green) and the proximal tubular marker lotus tetragonolobus lectin conjugated with Texas Red (LTL; red) in the kidneys from rats treated with a vehicle or angiotensin II (Ang II). Nuclei were stained with Hoechst33342 (blue, magnification 200 x). (C) Areas of green puncta were quantified by using image J. N ¼ 50 from 5 kidneys in each group. s.c.: subcutaneous. *p < 0.05 vs. Vehicle. Values are expressed as means ± SEM. Scale bar, 50 mm.

Article Snippet: Autophagosomes in renal tissues were visualized by immunostaining with anti-LC3 antibodies (Cell Signaling Technology, #12741) as previously reported.11 Hoechst33342 and lotus tetragonolobus lectin (LTL) conjugated with Texas Red (EY Laboratories, Inc., San Mateo, CA) were used for staining nuclei and proximal tubular cells, respectively.

Techniques: Marker, Staining

Journal: Journal of pharmacological sciences

Article Title: Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

doi: 10.1016/j.jphs.2020.12.001

Figure Lengend Snippet: Fig. 3. Time course of changes in levels of autophagosomes in proximal tubular cells before and 4 h and 24 h after I/R. (A) Representative images of (time course of) LC3 (green) and the proximal tubular marker lotus tetragonolobus lectin conjugated with Texas Red (LTL; red) in the kidneys of vehicle-treated and Ang II-treated rats. Renal tissues sampled before (Pre) and at 4 h (I/R-4h) and 24 h (I/R-24 h) after I/R were analyzed. Nuclei were stained with Hoechst33342 (magnification 200 x). (B) Areas of green puncta were quantified by using image J. N ¼ 50 from 5 kidneys in each group. *: p < 0.05 vs. Vehicle-Pre, y: p < 0.05 vs. Vehicle-4h. z: p < 0.05 vs. Ang II-Pre, x: p < 0.05 vs. Ang II-4h, Values are expressed as means ± SEM. Scale bar, 50 mm.

Article Snippet: Autophagosomes in renal tissues were visualized by immunostaining with anti-LC3 antibodies (Cell Signaling Technology, #12741) as previously reported.11 Hoechst33342 and lotus tetragonolobus lectin (LTL) conjugated with Texas Red (EY Laboratories, Inc., San Mateo, CA) were used for staining nuclei and proximal tubular cells, respectively.

Techniques: Marker, Staining

Journal: Journal of pharmacological sciences

Article Title: Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

doi: 10.1016/j.jphs.2020.12.001

Figure Lengend Snippet: Fig. 4. Time course of levels of LC3 (AeC), p62 (D and E) and beclin-1 (F and G) assessed by Western blotting in protocol 2. Pre: before ischemia-reperfusion, 4 h: 4 h after ischemia- reperfusion, 24 h: 24 h after ischemia-reperfusion. N ¼ 5 in each treatment. a.u.: arbitrary units. *: p < 0.05 vs Pre, y: p < 0.05 vs 4 h. Values are expressed as means ± SEM.

Article Snippet: Autophagosomes in renal tissues were visualized by immunostaining with anti-LC3 antibodies (Cell Signaling Technology, #12741) as previously reported.11 Hoechst33342 and lotus tetragonolobus lectin (LTL) conjugated with Texas Red (EY Laboratories, Inc., San Mateo, CA) were used for staining nuclei and proximal tubular cells, respectively.

Techniques: Western Blot

Journal: Journal of pharmacological sciences

Article Title: Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

doi: 10.1016/j.jphs.2020.12.001

Figure Lengend Snippet: Fig. 7. (A) Changes in protein expression levels of LC3-I and LC3-II caused by Ang II (1 mM for 4 h) in NRK-52E cells in vitro. N ¼ 11 in each treatment. *: p < 0.05 vs Vehicle. (B) Protein expression levels of LC3-I and LC3-II with or without Ang II or bafilomycin A1 (Baf). N ¼ 3 in each treatment. *p < 0.05 vs Ang II ()/Baf (). yp < 0.05 vs Ang II ()/Baf (þ). z: p < 0.05 vs Ang II (þ)/Baf (). (CeE) Effects of Ang II on levels of phospho-Thr172 and total AMPK (C), phospho-Ser317 and total ULK1 (D) and phospho-Ser473 and total Akt (E) in NRK-52E cells in vitro. N ¼ 11 in each treatment in (C) and (E). N ¼ 4 in each treatment in (D). a.u.: arbitrary units. *p < 0.05 vs Vehicle.

Article Snippet: Autophagosomes in renal tissues were visualized by immunostaining with anti-LC3 antibodies (Cell Signaling Technology, #12741) as previously reported.11 Hoechst33342 and lotus tetragonolobus lectin (LTL) conjugated with Texas Red (EY Laboratories, Inc., San Mateo, CA) were used for staining nuclei and proximal tubular cells, respectively.

Techniques: Expressing, In Vitro

Journal: Journal of pharmacological sciences

Article Title: Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

doi: 10.1016/j.jphs.2020.12.001

Figure Lengend Snippet: Fig. 8. Protein expression levels of LC3-II were significantly decreased by the AT1 receptor antagonist losartan (A) but not by the Mas receptor antagonist A779 (B) in NRK-52E cells in vitro. N ¼ 12 in each treatment in (A). N ¼ 10 in each treatment in (B). a.u.: arbitrary units. *p < 0.05 vs Vehicle.

Article Snippet: Autophagosomes in renal tissues were visualized by immunostaining with anti-LC3 antibodies (Cell Signaling Technology, #12741) as previously reported.11 Hoechst33342 and lotus tetragonolobus lectin (LTL) conjugated with Texas Red (EY Laboratories, Inc., San Mateo, CA) were used for staining nuclei and proximal tubular cells, respectively.

Techniques: Expressing, In Vitro

Journal: PLoS ONE

Article Title: Induction of Autophagy by a Novel Small Molecule Improves Aβ Pathology and Ameliorates Cognitive Deficits

doi: 10.1371/journal.pone.0065367

Figure Lengend Snippet: A, SH-SY5Y/LC3-GFP and MC65 cells were treated with 20 µM GTM-1 for 12 hrs. Next, the cell lysates were analyzed using western blotting with anti-LC3 and anti-β-tubulin (as a control). B, Left: SH-SY5Y and MC65 cells were treated with 20 µM GTM-1 for 6 hrs. Next, the cell lysates were analyzed using western blotting with anti-LC3 and anti-β-tubulin (as a control). Right: quantification of the Western blot images of the ratio between LC3-II and tubulin. C, MC65 cells were treated with vehicle control (1% DMSO) or GTM-1 (20 µM) for 8 hrs. The cells were then fixed with glutaraldehyde and prepared for EM analysis. Bar, 1∶11,000. Arrows indicate double and multi-membrane autophagosomic vesicles.

Article Snippet: Rabbit polyclonal antibody anti-LC3 and mouse monoclonal antibody anti-β-tubulin were obtained from Sigma.

Techniques: 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