Journal: Scientific Reports

Article Title: Novel 2-phenyloxypyrimidine derivative induces apoptosis and autophagy via inhibiting PI3K pathway and activating MAPK/ERK signaling in hepatocellular carcinoma cells

doi: 10.1038/s41598-018-29199-8

Figure Lengend Snippet: Compound E5 induced autophagy in HCC cells. ( A ) Representative fluorescent images of Bel7404 and HepG2 cells transfected with adenovirus expressing GFP-LC3 fusion protein and treated with E5 (20 μM) for 24 h. The images show GFP-LC3 dots formation in E5-treated cells. ( B ) and ( C ) Western blotting of the expression of LC3-I, LC3-II and Atg5. ( D ) Western blotting of the expression of LC3 after treatment with 20 μM E5 with or without CQ (10 μM) for 24 h in Bel7404 and HepG2 cells. ( E ) Western bloting of siRNA mediated Atg5 knockdown (Left). Knockdown of Atg5 rescued HCC cells from E5-reduced cell viability as assessed by MTT assay (Right). ( F ) Autophagy inhibitor 3-MA rescued HCC cells from E5-reduced cell viability. HCC cells were exposed to 3-MA (2 mM) with or without E5 (20 μM) for 24 h, cell viability was measured by MTT assay. The viability of untreated cells was considered 100%. The data were presented as mean ± SD. * P < 0.05; ** P < 0.01 compared to DMSO control; one-way ANOVA, post-hoc intergroup comparisons, Tukey’s test. Original gel images are presented in Supplementary Figs – .

Article Snippet: Antibody to LC3 (#AL221) was purchased from Beyotime Institute of Biotechnology (Shanghai, China).

Techniques: Transfection, Expressing, Western Blot, Knockdown, MTT Assay, Control

Journal: Scientific Reports

Article Title: Novel 2-phenyloxypyrimidine derivative induces apoptosis and autophagy via inhibiting PI3K pathway and activating MAPK/ERK signaling in hepatocellular carcinoma cells

doi: 10.1038/s41598-018-29199-8

Figure Lengend Snippet: Compound E5 inhibited PDGFRα/PI3K/Akt/mTOR pathway and activateed MAPK/ERK pathway in HCC cells. ( A ) Bel7404 cells and HepG-2 cells were treated with incremental concentrations of E5 for 24 h. The protein levels of PDGFRα, PI3K/AKT/mTOR, MEK/ERK and their phosphorylation status were examined by Western blotting. ( B ) MEK/ERK inhibitor U0126 rescued HCC cells from E5-reduced cell viability and inhibited the conversion of LC3-I to LC3-II. HCC cells were exposed to U0126 (10 μM) with or without E5 (20 μM) for 24 h, cell viability was measured by MTT assay, and the expression of ERK and LC-3 was examined by Western blot. The data were presented as mean ± SD. * P < 0.05; ** P < 0.01 compared to DMSO control; one-way ANOVA, post-hoc intergroup comparisons, Tukey’s test. Original gel images are presented in Supplementary Figs and . ( C ) Schematic illustration of possible pathways involved in the apoptosis and autophagy by compound E5 in HCC cells.

Article Snippet: Antibody to LC3 (#AL221) was purchased from Beyotime Institute of Biotechnology (Shanghai, China).

Techniques: Phospho-proteomics, Western Blot, MTT Assay, Expressing, Control

Journal: Ecotoxicology and environmental safety

Article Title: Regulation of Parkin in Cr (VI)-induced mitophagy in chicken hepatocytes.

doi: 10.1016/j.ecoenv.2022.114315

Figure Lengend Snippet: Fig. 2. (A–D) The p62, LC3, and Parkin levels were detected using Western blot and quantitative analysis. (E) Protein expression of PINK1 was detected by ELISA. Data are presented as mean ± SEM (n = 3) (P < 0.05 vs. control). (F–G) Mitochondrial membrane potential depolarization levels were detected using flow cytometer and quantitative analysis. Data are presented as mean ± SEM (n = 3). Means that do not share common letters (a–c) differ significantly (P < 0.05 vs. control).

Article Snippet: Sealing solution (5 % skimmed milk powder) was blocked for 2 h and incubated overnight with the following antibodies: LC3 B (Beyotime, AL221), p62 (protein test, 18420-1-AP), Parkin (Proteintech, 14060-1-AP) and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Control, Membrane, Flow Cytometry

Journal: Ecotoxicology and environmental safety

Article Title: Regulation of Parkin in Cr (VI)-induced mitophagy in chicken hepatocytes.

doi: 10.1016/j.ecoenv.2022.114315

Figure Lengend Snippet: Fig. 4. (A–D) The p62, LC3, and Parkin levels were detected using Western blot and quantitative analysis. (E) Protein expression was detected by ELISA. Data are presented as mean ± SEM (n = 3) (P < 0.05 vs. control). (F–G) Mitochondrial membrane potential depolarization levels were detected using flow cytometer and quantitative analysis. Data are presented as mean ± SEM (n = 3) (P < 0.05 vs. control).

Article Snippet: Sealing solution (5 % skimmed milk powder) was blocked for 2 h and incubated overnight with the following antibodies: LC3 B (Beyotime, AL221), p62 (protein test, 18420-1-AP), Parkin (Proteintech, 14060-1-AP) and GAPDH (Proteintech, 60004-1-Ig).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Control, Membrane, Flow Cytometry

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, Negative Control, Small Interfering RNA

Journal: Biologics : Targets & Therapy

Article Title: Autophagy regulates the stemness of cervical cancer stem cells

doi: 10.2147/BTT.S134920

Figure Lengend Snippet: The characteristics of the cervical cancer tumorspheres. ( A ) Tumorsphere cultured in serum-free medium for 10–12 days. Magnification ×40 (left) and ×100 (right). (B ) Immunofluorescence detected the expression of Oct4 and stem cell marker CD133 in tumor sphere cells. ( C ) The tumor sphere cells were further induced with lipid cells. ( D ) After 14–21 days of induction, the lipid drop-like oil “O” positive cells were found. Magnification ×100. Scale bars =100 μm.

Article Snippet: The antibody dilutions were as follows: anti-LC3 rabbit mAb (Beyotime, AL221, 1:500); anti-Beclin 1 rabbit mAb (CS, 3495, 1:250); anti-phospho-mTOR rabbit mAb (CS, 5536, 1:250); anti-phospho-4EBP1 rabbit mAb (CS, 2855P, 1:250); Oct-4A rabbit mAb (CS, 2840P, 1:250); Sox2 rabbit mAb (CS, 3579P, 1:250); CD133 rabbit mAb (Sigma-Aldrich, SAB4300882, 1:250) and GAPDH mouse monoclonal antibody (Beyotime, AF0006, 1:200).

Techniques: Cell Culture, Immunofluorescence, Expressing, Marker

Journal: Biologics : Targets & Therapy

Article Title: Autophagy regulates the stemness of cervical cancer stem cells

doi: 10.2147/BTT.S134920

Figure Lengend Snippet: Effects of autophagy on regulating proteins in cervical cancer stem cells. ( A ) Western blot detected the expression levels of pluripotency-associated proteins Oct4, Sox2, and CD133 in both adherent and tumorsphere HeLa cells. ( B ) Western blot detected the effect of HBSS on the level of pluripotency-associated proteins Oct4, Sox2 and CD133 in both adherent and tumorsphere HeLa cells at 0, 4 and 8 h. ( C ) Western blot detected the mTOR-downstream markers p-mTOR and p-4EBP when cells were treated with HBSS for 0, 2, 4 and 8 h.

Article Snippet: The antibody dilutions were as follows: anti-LC3 rabbit mAb (Beyotime, AL221, 1:500); anti-Beclin 1 rabbit mAb (CS, 3495, 1:250); anti-phospho-mTOR rabbit mAb (CS, 5536, 1:250); anti-phospho-4EBP1 rabbit mAb (CS, 2855P, 1:250); Oct-4A rabbit mAb (CS, 2840P, 1:250); Sox2 rabbit mAb (CS, 3579P, 1:250); CD133 rabbit mAb (Sigma-Aldrich, SAB4300882, 1:250) and GAPDH mouse monoclonal antibody (Beyotime, AF0006, 1:200).

Techniques: Western Blot, Expressing

Journal: Biologics : Targets & Therapy

Article Title: Autophagy regulates the stemness of cervical cancer stem cells

doi: 10.2147/BTT.S134920

Figure Lengend Snippet: The effect of autophagy inhibitor 3-methyladenine (3-MA) on cervical cancer cells. ( A ) Acridine orange staining detected autophagic flux in cancer stem cells and adherent cells with and without 3-MA treatment. The orange fluorescence represents autophagosomes. Magnification ×100. ( B ) xCELLigence RTCA (top) and Transwell (bottom) detected the effects of 3-MA on HeLa cell proliferation and invasion. ( C ) Western blot detected the effected of 3-MA on the level of PA proteins Oct4, Sox2 and CD133 in both adherent and tumorsphere HeLa cells. # P <0.05. Scale bar =100 μm.

Article Snippet: The antibody dilutions were as follows: anti-LC3 rabbit mAb (Beyotime, AL221, 1:500); anti-Beclin 1 rabbit mAb (CS, 3495, 1:250); anti-phospho-mTOR rabbit mAb (CS, 5536, 1:250); anti-phospho-4EBP1 rabbit mAb (CS, 2855P, 1:250); Oct-4A rabbit mAb (CS, 2840P, 1:250); Sox2 rabbit mAb (CS, 3579P, 1:250); CD133 rabbit mAb (Sigma-Aldrich, SAB4300882, 1:250) and GAPDH mouse monoclonal antibody (Beyotime, AF0006, 1:200).

Techniques: Staining, Fluorescence, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

doi: 10.1111/jcmm.13328

Figure Lengend Snippet: A TLR3 agonist polyinosinic‐polycytidylic acid (poly(I:C)) induced autophagy in cultured cardiomyocytes through a TRIF‐dependent pathway. ( A ) Poly(I:C) increased autophagy markers in cultured H9c2 rat ventricular cells. ( B ) Poly(I:C) stimulated autophagosome formation but did not affect autophagic flux. Primary cultured neonatal rat ventricular myocytes (NRVMs) were transfected with a tandem mRFP‐GFP‐LC3 adenovirus for 24 hrs, followed by treatment with poly(I:C) (100 μg/ml, 4 hrs). Autophagosomes and autolysosomes were, respectively, visualized as yellow‐ and red‐only punctas under a confocal microscope. ( C ) An autophagic flux inhibitor chloroquine (CQ) induced accumulations of LC3‐II and p62/SQSTM1 proteins in H9c2 myocytes receiving poly(I:C) (100 μg/ml, 4 hrs). CQ was applied at 10 μM, immediately prior to poly(I:C). ( D ) Effects of indicated siRNA on poly(I:C)‐induced changes in autophagy markers in NRVMs. All the siRNAs were transfected at 50 nM for 48 hrs, and poly(I:C) was added at 100 μg/ml for 4 hrs before cell harvest. Negative control (NC) siRNA served as control. RNAiMAX transfection reagent was used in all the siRNA experiments. The upper panel shows the knockdown effects of siRNAs, and the lower panel shows representative Western blot images (presented from four independent experiments) and densitometry quantitative data (normalized into ‘fold of vehicle group’). All quantitative data are expressed as means ± S.D. a P < 0.05, A P < 0.01 versus vehicle; b P < 0.05, B P < 0.01 versus poly(I:C).

Article Snippet: The primary antibodies against TLR3 (Cat. NB100‐56571), MyD88 (Cat. NBP1‐19785) and Trif (Cat. NB120‐13810) were purchased from Novus Biologicals, LLC, Littleton, CO, USA; the anti‐LC3 antibody (Cat. AL221) was from Beyotime Institute of Biotechnology, Jiangsu, China; the anti‐beclin‐1 antibody (Cat. 11306‐1‐AP) was from Proteintech Group, Inc., Rosemont, IL, USA; and the anti‐p62 antibody (Cat. 5114) was from Cell Signalling Technology, Inc., Danvers, MA, USA.

Techniques: Cell Culture, Transfection, Microscopy, Negative Control, Control, Knockdown, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

doi: 10.1111/jcmm.13328

Figure Lengend Snippet: Autophagy induction abolished the protection of TLR3‐KO against MI. An autophagy inducer rapamycin (Rapa, 2 mg/kg/day) or an autophagic flux inhibitor chloroquine (CQ, 50 mg/kg/day) was daily intraperitoneally injected for 2 weeks, starting from day 1 after surgery. Normal saline (NS) was injected as control. Measurements were taken at 2 weeks. ( A ) Representative Western blot images and quantitative data of LC3 and p62 proteins in infarct tissue. Rapamycin increased LC3‐II in all the groups, suggesting successful induction of autophagy. CQ (blue bars) induced similar accumulations of LC3‐II and p62 in WT and TLR3‐KO myocardium, suggesting that autophagy flux was comparable between the two groups. ( B ) Representative coronal‐sectional images of Masson's trichrome staining and infarct size of post‐infarct hearts. ( C ) Fractional shortening (FS, %) of the left ventricle. All data are means ± S.D. n = 4–6 mice/group. A P < 0.01 versus respective ‘sham+NS’; b P < 0.05, B P < 0.01 versus respective ‘MI+NS’.

Article Snippet: The primary antibodies against TLR3 (Cat. NB100‐56571), MyD88 (Cat. NBP1‐19785) and Trif (Cat. NB120‐13810) were purchased from Novus Biologicals, LLC, Littleton, CO, USA; the anti‐LC3 antibody (Cat. AL221) was from Beyotime Institute of Biotechnology, Jiangsu, China; the anti‐beclin‐1 antibody (Cat. 11306‐1‐AP) was from Proteintech Group, Inc., Rosemont, IL, USA; and the anti‐p62 antibody (Cat. 5114) was from Cell Signalling Technology, Inc., Danvers, MA, USA.

Techniques: Injection, Saline, Control, Western Blot, Staining