Journal: Frontiers in Pharmacology

Article Title: Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson’s Disease Model

doi: 10.3389/fphar.2020.618787

Figure Lengend Snippet: BBR mitigates autophagic impairment in MPTP-induced mice. (A) The representative immunohistochemical staining for MAP1LC3B in SN. (B) The number of MAP1LC3B positive cells in SN. Representative western blot bands (C) and the statistical graph (D) of MAP1LC3B and BECN1 in SN. (E) Transmission electron microscopy shown with autophagosomes (yellow arrows) in the SN. Data were expressed as the mean ± SD ( n = 6). * p < 0.05 compared with control group, # p < 0.05 compared with MPTP group, + p < 0.05 compared with MPTP + BBR group. MAP1LC3B, microtubule associated protein 1 light chain 3 beta; SN, substantia nigra; BECN1, beclin 1.

Article Snippet: The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026).

Techniques: Immunohistochemical staining, Staining, Western Blot, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson’s Disease Model

doi: 10.3389/fphar.2020.618787

Figure Lengend Snippet: BBR enhances autophagic activity in MPP + -treated BV2 cells. The representative immunofluorescent staining (A) and puncta (B) of MAP1LC3B in BV2 cells treated with MPP + at 200 μM and BBR at the concentration of 12.5, 25, and 50 μM (20 cells were analyzed per group for MAP1LC3B puncta counting). Representative western blots (C) and the statistical graph (D) of MAP1LC3B and BECN1 in BV2 cells treated with MPP + at 200 μM and BBR at the concentration of 12.5, 25, and 50 μM. The representative double-immunofluorescent staining (E) and puncta (E) of MAP1LC3B in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM (20 cells were analyzed per group for MAP1LC3B puncta counting). The representative monodansylcadaverine staining (G) and statistical graph (H) of autophagic vesicles in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM. Representative western blot bands (I) and the statistical graph (J) of MAP1LC3B and BECN1 in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM. Data were expressed as the mean ± SD ( n = 3). * p < 0.05 compared with untreated group, # p < 0.05 compared with MPP + group, + p < 0.05 compared with MPP + + BBR group. MAP1LC3B, microtubule associated protein 1 light chain 3 beta; BECN1, beclin 1.

Article Snippet: The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026).

Techniques: Activity Assay, Staining, Concentration Assay, Western Blot

Journal: Molecular Medicine Reports

Article Title: Ginsenoside metabolite 20(S)-protopanaxadiol promotes neural stem cell transition from a state of proliferation to differentiation by inducing autophagy and cell cycle arrest

doi: 10.3892/mmr.2020.11081

Figure Lengend Snippet: PPD promotes NSC differentiation by inducing autophagy. (A) NSCs were treated with 20 µM PPD for 24, 48 or 72 h. The indicated protein levels were analyzed by western blotting. (B) Expression of tubulin-β3 (neuronal marker) was significantly increased in NSCs following treatment with 20 µM PPD. (C) In total, 20 µM PPD treatment for 48 h significantly increased the expression of LC3II compared with the respective control group, (D) p62 expression, which serves as another indicator of autophagy, was increased at 24 h in the PPD treatment group, which was significantly earlier than that at 48 h in the control group. *P<0.05 and **P<0.01 vs. DMSO group. LC3, light chain 3; PPD, 20(S)-protopanaxadiol.

Article Snippet: Subsequently, cells were incubated with the following primary antibodies overnight at 4°C: Mouse monoclonal anti-BrdU (1:100; Sigma-Aldrich; Merck KGaA; cat. no. NA61), Rabbit monoclonal anti-tubulin-β3 (1:100; Abcam; cat. no. ab18207) and Rabbit polyclonal anti-LC3 (1:100; Bioworld Technology, Inc.; cat. no. AP0762).

Techniques: Western Blot, Expressing, Marker

Journal: Molecular Medicine Reports

Article Title: Ginsenoside metabolite 20(S)-protopanaxadiol promotes neural stem cell transition from a state of proliferation to differentiation by inducing autophagy and cell cycle arrest

doi: 10.3892/mmr.2020.11081

Figure Lengend Snippet: LC3 punctae formation and autophagic vacuoles. (A) Immunofluorescence images of autophagy following treatment with 20 µM PPD, as indicated by LC3 immunostaining (scale bar, 100 µm). (B) The percentage of cells with LC3 punctate. (C) Transmission electron microscopy images displayed autophagosome formation in NSCs following treatment with 20 µM PPD for 72 h, the arrows indicate autophagic vacuoles. scale bar, 2 µm. (D) The percentage of autophagic vacuoles area in each per field. *P<0.05 vs. the DMSO group. LC3, light chain 3; PD, 20(S)-protopanaxadiol.

Article Snippet: Subsequently, cells were incubated with the following primary antibodies overnight at 4°C: Mouse monoclonal anti-BrdU (1:100; Sigma-Aldrich; Merck KGaA; cat. no. NA61), Rabbit monoclonal anti-tubulin-β3 (1:100; Abcam; cat. no. ab18207) and Rabbit polyclonal anti-LC3 (1:100; Bioworld Technology, Inc.; cat. no. AP0762).

Techniques: Immunofluorescence, Immunostaining, Transmission Assay, Electron Microscopy

Journal: International Journal of Biological Sciences

Article Title: Slit2/Robo1 Mitigates DSS-induced Ulcerative Colitis by Activating Autophagy in Intestinal Stem Cell

doi: 10.7150/ijbs.42331

Figure Lengend Snippet: Expression of the autophagy proteins in DSS-induced mice. The coexpression of Lgr5 + colonic stem cells and the autophagy protein LC3 in (A) WT Slit and Slit2-Tg mice and (B) WT Robo1/2 and Robo1/2 +/- mice. Lgr5 (green) and LC3 (red) expression in the colonic epithelium. Sections were counterstained with DAPI (blue). (n=5 in each group; scale bar = 50 μm); (C) the photograph of the crypt isolated from colon tissue (scale bar= 200μm); (D) protein expression of LC3II/I in (D) WT Slit and Slit2-Tg mice and (F) WT Robo1/2 and Robo1/2 +/- mice (n=4); The expression of P62 in (E) WT Slit and Slit2-Tg mice and (G) WT Robo1/2 and Robo1/2 +/- mice (n=3-4 in each group). Detection of β-actin served as a loading control. Quantification of bands is expressed as density ratio of indicated protein/β-actin (A.U.); data are present as means ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Rabbit anti-mouse LC3 polyclonal antibody (1:500, ABclonal), rabbit anti-mouse p62 polyclonal antibody (1:500, Affinity Biosciences), and mouse anti-mouse β-actin (1:10000, Boster) were added and incubated at 4°C overnight.

Techniques: Expressing, Isolation, Control