Fig 1: High-dose doxorubicin specifically activated autophagy flux in DR-BC cells, instead of their parental DS-BC cells. a-f By conducting Western Blot analysis, we found that doxorubicin specifically increased LC3B-II/I ratio, and decreased p62 expression levels in DR-BC cells (full-length blots/gels are presented in Supplementary Fig. S5A-B). g, h Electronic microscope (EM) was used to observe the autophagosomes (indicated by the red arrows), and the magnificence of the images were 5000 ×. i-l The relative levels of LC3B-II/I ratio was determined by Western Blot analysis in DR-BC cells (full-length blots/gels are presented in Supplementary Fig. S5C-D). Each experiment repeated at least 3 times, and *P < 0.05 was regarded as statistical significance
Fig 2: High-dose doxorubicin activated autophagy flux in DR-BC cells through regulating AMPK-ULK1 pathway, examined by Western Blot analysis. a, b High-dose doxorubicin specifically increased the expression levels of p-AMPLK and p-ULK1 to activate AMPK-ULK1 pathway in DR-BC cells, instead of DS-BC cells. The AMPK-ULK1 pathway was successfully blocked by its inhibitor, c, d compound C and e, f SBI-0206965. g, h Blockage of AMPK-ULK1 pathway decreased LC3B-II/I ratio, and increased p62 expression levels to reverse doxorubicin induced autophagy in DR-BC cells (full-length blots/gels are presented in Supplementary Fig. S6A-H). Each experiment repeated at least 3 times, and *P < 0.05 was regarded as statistical significance
Fig 3: SJP inhibits excessive autophagy through the ALKBH5/GSK3β pathway in H/R-induced H9c2 cells (n = 3). a and b H9c2 cardiomyocytes were transfected with adenoviruses harboring tandem fluorescent mRFP-GFP-LC3 (Ad-LC3-H9c2s) for 24 h and subjected to various treatments (bar = 50 μm). c and d Numbers of autophagosomes determined by TEM (× 40,000). e and f Apoptotic rates of H/R-treated H9c2 cells assessed by the Annexin V/PI assay. g and h Protein levels of p-GSK3β/GSK3β, p-mTOR/mTOR, LC3B (II/I), Atg5, and p62 in H/R-treated H9c2 cells evaluated by western blot. Data are mean ± SEM. *P < 0.05 versus shRNA-ALKBH5 + H/R group; #P < 0.05 versus shRNA-ALKBH5 + GSK3β-inhibitor + H/R group
Fig 4: SJP inhibits autophagic activity through the ALKBH5 and GSK3β/mTOR pathway in gene knockout experiments following H/R (n = 3). a and b H9c2 cardiomyocytes underwent transfection with adenovirus carrying tandem fluorescent mRFP-GFP-LC3 (Ad-LC3-H9c2s) for 24 h and various treatments (bar = 50 μm). c and d The protein amounts of p-GSK3β/GSK3β, p-mTOR/mTOR, LC3B (II/I), Beclin-1, and p62 in H/R-treated H9c2 cells by western blot. e and f Apoptotic rates of cardiomyocytes in H/R-induced H9c2 cells assessed by the Annexin V/PI assay. Data are mean ± SEM. *P < 0.05 versus shRNA-Ctrl + H/R group; # P < 0.05 versus shRNA-ALKBH5 + H/R group; &P < 0.05 versus shRNA-GSK3β + H/R group
Fig 5: Evidence of autophagy observed in U87MG cells treated with PBM. (A) Total protein lysate is collected immediately or 1–5 h after PBM. (B) Cells exposed to rotenone for 24 h and then treated with PBM. The total protein lysate is collected immediately or 1–5 h after PBM. (C) Representative fluorescence images of cells exposed to PBM and rotenone and immunolabeled with antibodies against cytochrome C (red), giantin (blue), and LC3B (green). Cell nuclei are labeled with DAPI (blue and pink). The representation of each channel is shown in (D). White arrows mark the position of the Golgi apparatus. Giantin and DAPI images are subjected to postprocessing (subtraction of DAPI image from Giantin + DAPI image) to highlight the Golgi apparatus in yellow.
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