Journal: Cell & Bioscience

Article Title: Ubiquitinated AIF is a major mediator of hypoxia-induced mitochondrial dysfunction and pulmonary artery smooth muscle cell proliferation

doi: 10.1186/s13578-022-00744-3

Figure Lengend Snippet: Effects of hypoxia and AIF on mitophagy and autophagy in PASMCs. A AIF influenced mitochondrial function by obstructing Tom20 and Lamp2a colocalization, as indicated by the decreased yellow area. Scale bars: 50 μm (n = 4). B PASMCs were exposed to hypoxia for 24 h, and the expression of Pink and Parkin was evaluated by Western blotting (n = 5). C AIF overexpression mitigated the alteration of mitophagy in PASMCs as determined by Mitophagy Keima-Red plasmid transfection. The number of mitochondrial autophagosomes (yellow dots) was calculated (n = 5). Scale bars: 50 μm. D The expression of LC3B-II, P62 and ATG5/7 was evaluated by Western blotting (n = 5). E Autophagic flux and the formation of autophagosomes were detected (n = 6). Scale bars: 50 μm. F Representative electron micrograph of cells after Nor and Hyp treatment. Asterisks represent autophagosomes and arrows represent autolysosomes. Scale bars: 500 nm (n = 3). All data are presented as the means ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; Nor normoxia, Hyp hypoxia, NC negative control

Article Snippet: Antibodies against Cyclin A, Cyclin D, PCNA, UB, P62, ATG5 and ATG7 (Catalog numbers BM1582, BM4272, BM0104, BM4359, BA2849, BA3525 and BA3527) were obtained from Boster (Wuhan, China).

Techniques: Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Standard Deviation, Negative Control

Journal: Oncology Letters

Article Title: Endocan silencing induces programmed cell death in hepatocarcinoma

doi: 10.3892/ol.2017.6857

Figure Lengend Snippet: The levels of LC3, ATG5, ATG7, DRAM and Beclin-1 protein expression were measured following transfection with endocan siRNA and normalized to β-actin. (A) Representative blot and (B) quantification of autophagy-associated protein expression. The expression levels of all the autophagy-associated proteins were significantly increased in endocan siRNA-treated cells compared with those in the NC and control groups. *P<0.05 in siRNA groups vs. NC groups. NC, negative control; siRNA, small interfering RNA; LC3, microtubule associated protein 1 light chain 3 α; ATG, autophagy related; DRAM, DNA damage regulated autophagy modulator 1.

Article Snippet: The primary antibodies were as follows: Endocan antibody (sc-515304; Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight; LC3 antibody (AL221; Beyotime Institute of Biotechnology; 1:500) at 4°C overnight; ATG5 antibody (PA2260; Boster Biological Technology, Pleasanton, CA, USA; 1:400,) at 4°C overnight; ATG7 antibody (PB9479; Boster Biological Technology; 1:400) at 4°C overnight; DRAM antibody (bs-4233R, BIOSS, Beijing, China; 1:500) at 4°C overnight; Beclin-1 antibody (AB123; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight and NF-κB p65 antibody (AF0246; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight.

Techniques: Expressing, Transfection, Control, Negative Control, Small Interfering RNA

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-β1/SMAD pathway

doi: 10.1186/s13287-022-02996-9

Figure Lengend Snippet: Hypoxic hBMSCs promote HaCaT cell autophagy, proliferation and migration in a paracrine manner. a Western blotting and b quantitative analysis were used to analyze the expression levels of ATG5, ATG7, LC3B-I/II, BECN1 and SQSTM1 in HaCaT cells after treatment with different conditioned media for 24 h. c MTS and d EdU assays were used to assess the proliferation of HaCaT cells after 24 h of treatment with different CM. Bar, 100. e Scratch assays and f quantitative analysis were performed to detect the migration of HaCaT cells after the cells were treated with different conditioned media for 24 h. Bar, 200 μm. Mean ± SEM. n = 3. * P < 0.05, ** P < 0.01

Article Snippet: The following primary antibodies were used: HIF-1α (1:200, Proteintech, 20960-1-AP), ATG5 (1:200, HUABIO, ET1611-38), ATG7 (1:200, HUABIO, ET1610-53), K14 (1:200, Proteintech, 60320-1-Ig), and SMAD4 (1:200, CST, #8685).

Techniques: Migration, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-β1/SMAD pathway

doi: 10.1186/s13287-022-02996-9

Figure Lengend Snippet: The autophagic effect of hypoxic hBMSCs promotes HaCaT cell proliferation and migration. a qRT-PCR and b , c Western blotting was used to measure the mRNA and protein levels of ATG5 and ATG7 to evaluate the efficiency of siATG5 and siATG7 in HaCaT cells at 24 h after hyCM administration. d Western blotting and e quantitative analysis were used to analyze the expression levels of LC3B-I/II and SQSTM1 in control HaCaT cells and siATG5 and siATG7 HaCaT cells after the cells were treated with different conditioned media for 24 h. f MTS and g EdU assays were used to assess the proliferation of these HaCaT cells after 24 h of treatment with different CM. Bar, 100. h Scratch assays and i quantitative analysis were performed to detect the migration of HaCaT cells after treatment with different conditioned media for 24 h. Bar, 200 μm. Mean ± SEM. n = 3. * P < 0.05, ** P < 0.01

Article Snippet: The following primary antibodies were used: HIF-1α (1:200, Proteintech, 20960-1-AP), ATG5 (1:200, HUABIO, ET1611-38), ATG7 (1:200, HUABIO, ET1610-53), K14 (1:200, Proteintech, 60320-1-Ig), and SMAD4 (1:200, CST, #8685).

Techniques: Migration, Quantitative RT-PCR, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-β1/SMAD pathway

doi: 10.1186/s13287-022-02996-9

Figure Lengend Snippet: hBMSC-derived TGF-β1 contributes to the activation of HaCaT cell autophagy by hypoxic hBMSCs. a Western blotting and b quantitative analysis were used to measure the protein level of TGF-β1 to evaluate the efficiency of siTGF-β1 in hBMSCs after 24 h of hypoxia exposure. c ELISA was performed to measure the protein level of secreted TGF-β1 in the CM from control hBMSCs and siTGF-β1 hBMSCs after 24 h of hypoxia. d Western blotting and e quantitative analysis were used to analyze the expression levels of ATG5, ATG7, LC3B-I/II, BECN1 and SQSTM1 in HaCaT cells after 24 h of exposure to CM from control hy-hBMSCs and siTGF-β1 hy-hBMSCs. Mean ± SEM. n = 3. * P < 0.05, ** P < 0.01

Article Snippet: The following primary antibodies were used: HIF-1α (1:200, Proteintech, 20960-1-AP), ATG5 (1:200, HUABIO, ET1611-38), ATG7 (1:200, HUABIO, ET1610-53), K14 (1:200, Proteintech, 60320-1-Ig), and SMAD4 (1:200, CST, #8685).

Techniques: Derivative Assay, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-β1/SMAD pathway

doi: 10.1186/s13287-022-02996-9

Figure Lengend Snippet: Knockdown of TGF-β1 in mBMSCs attenuates the therapeutic effects on diabetic wounds. a Representative pictures and b quantitation of the healing time of control mBMSC-treated and siTGF-β1 mBMSC-treated and untreated diabetic wounds after an 8-mm primary biopsy. n = 6. c Representative H&E-stained images of the wounds on day 7. The epithelium is marked with a dotted line. Bars, 50 μm. d Western blotting and e quantitative analysis were used to analyze the expression levels of Smad2, p-Smand2, atg5, atg7, lc3b-I/II, beclin1 and p62 in the wounds on day 7 after wounding. f Representative images of wounded (day 7) skin stained to show the expression of atg5 or atg7 (green) and K14 (red) in the wounds (blue). Bar, 100 μm. Mean ± SEM. n = 3 (in addition to the quantitation of healing time). * P < 0.05, ** P < 0.01

Article Snippet: The following primary antibodies were used: HIF-1α (1:200, Proteintech, 20960-1-AP), ATG5 (1:200, HUABIO, ET1611-38), ATG7 (1:200, HUABIO, ET1610-53), K14 (1:200, Proteintech, 60320-1-Ig), and SMAD4 (1:200, CST, #8685).

Techniques: Quantitation Assay, Staining, Western Blot, Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Molecular cloning and expression analyses of porcine MAP1LC3A in the granulosa cells of normal and miniature pig

doi: 10.1186/1477-7827-11-8

Figure Lengend Snippet: Primers used for the full-length cloning and expression of the porcine MAP1LC3A gene

Article Snippet: The membrane was then incubated for 2 h with anti-rabbit MAP1LC3A monoclonal antibody (diluted 1:1000; Abcam, MA), anti-rabbit ATG5 polyclonal antibody (diluted 1:1000; Abfrontier, Seoul, Kor).

Techniques: Clone Assay, Expressing, Sequencing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Molecular cloning and expression analyses of porcine MAP1LC3A in the granulosa cells of normal and miniature pig

doi: 10.1186/1477-7827-11-8

Figure Lengend Snippet: Effect of rapamycin treatment on the expression of MAP1LC3A and ATG5 protein expression in cultured granulosa cells. A. Immunofluorescence staining of cultured granulosa cells. B. Western blot analysis of the porcine MAP1LC3A protein. C. Real-time PCR of porcine MAP1LC3A mRNA. Data represent the mean ± standard error of 3 individual experiments (p < 0.05).

Article Snippet: The membrane was then incubated for 2 h with anti-rabbit MAP1LC3A monoclonal antibody (diluted 1:1000; Abcam, MA), anti-rabbit ATG5 polyclonal antibody (diluted 1:1000; Abfrontier, Seoul, Kor).

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Western Blot, Real-time Polymerase Chain Reaction