Journal: Cancer Biology & Medicine

Article Title: VDAC1 protein derived from extracellular vesicles promotes paclitaxel resistance in gastric cancer through autophagy and mitophagy

doi: 10.20892/j.issn.2095-3941.2025.0360

Figure Lengend Snippet: Proteomic landscapes were established in EVs derived from PTX-resistant cells. (A) Schematic diagram of the proteomics design. (B) Volcano diagram analysis of differentially expressed proteins in PTX-sensitive (AGS) and -resistant cells (AGSR) and the secreted EVs (AGS-EVs or AGSR-EVs). (C) Venn diagram analysis of the differentially expressed proteins between the cell (AGSR) and the secreted EVs (AGSR-EVs). (D) Heatmap analysis of the overlapping proteins in cells (AGSR) and EVs (AGSR-EVs). (E) KEGG analysis of overlapped proteins in cells (AGSR) and EVs (AGSR-EVs). (F) The expression trends of the top 10 overlapping proteins in cells (AGSR) and EVs (AGSR-EVs) were analyzed using scatter plots. (G) The levels of VDAC1, HTRA1, and STEAP4 expression in cells (AGSR and HGC-27R) and EVs (AGSR-EVs and HGC-27R-EVs) were detected by western blot analysis. EV, extracellular vesicle; HTRA1, HtrA serine peptidase 1; PTX, paclitaxel; STEAP4, Six Transmembrane Epithelial Antigen of Prostate 4; VDAC1, voltage-dependent anion channel protein 1.

Article Snippet: The primary antibodies used in western blots were as follows: β-actin (1:1000, T002; Affinity, Cincinnati, OH, USA); GAPDH (1:5000, 104941-AP; Proteintech, Wuhan, China); P-gp (1:3000, 22336-1-AP; Proteintech); CD9 (1:1000, sc-13118; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HSP90 (1:5000, 13171-1-AP; Proteintech); calnexin (1:1000; c-23954; Santa Cruz Biotechnology, Inc. USA); TSG101 (1:1000, c-7964; Santa Cruz Biotechnology, Inc.); VDAC1 (1:1000, PTM-6157; PTM Biolabs, Chicago, IL, USA); p62 (1:1000, #5114; Cell Signaling Technology, Danvers, Massachusetts, USA); LC3B (1:1000, L7543; Sigma); ULK1 (1:1000, 20986-1-AP; Proteintech); p-ULK1 (1:1000, #14202; Cell Signaling Technology); AMPK (1:1000, #5831; Cell Signaling Technology); p-AMPK (1:1000, #2535; Cell Signaling Technology); mTOR (1:5000, 20657-1-AP; Proteintech); p-mTOR (1:2000, 67778-1-Ig; Proteintech); p70S6K (1:2000, 14485-1-AP; Proteintech); p-p70S6K (1:1000, #AF3228; Affinity); ATG5 (1:1000, 10181-2-AP; Proteintech); ATG7 (1:1000, 10088-2-AP; Proteintech); HTRA1 (1:1000, 55011-1-AP, Proteintech); and STEAP4 (1:1000, 11944-1-AP; Proteintech).

Techniques: Derivative Assay, Expressing, Western Blot

Journal:

Article Title: Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization

doi: 10.1093/emboj/cdg423

Figure Lengend Snippet: Fig. 1. Recombinant pro-apoptotic Bcl-2 members induce the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not that of EndoG and AIF from isolated mitochondria. (A) Mitochondria isolated from HeLa cells were incubated for 30 min at 30°C with different concentrations (nM) of recombinant oligomeric Bax. Mitochondrial pellets and supernatant fractions were separated by SDS–PAGE (Tricine Gel), and their respective cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF contents were analyzed by western blotting. (B) Mitochondria isolated from HeLa cells were incubated with 200 nM oligomeric recombinant Bax or with control buffer (none) at 30°C and the mitochondrial pellet and supernatant were analyzed at different time points (min), as in (A). (C) Mitochondria isolated from HeLa cells were incubated for 15 min with different concentrations (nM) of recombinant tBid and the mitochondrial pellet and supernatant were analyzed as in (A). In (A), (B) and (C), equal loading of the mitochondrial pellet was controlled using VDAC. The asterisks in (A), (B) and (C) indicate an additional band due to the previous VDAC detection.

Article Snippet: Antibodies used in immunoblotting were as follows: anti-cytochrome c mAb (clone 7H8.2C12, Pharmingen) (1/2000), rabbit polyclonal anti-Endo G (Pro-Sci Inc.) (1/1000), rabbit polyclonal anti-Smac/Diablo (Pro-Sci Inc.) (1/1000), a purified rabbit polyclonal anti-HtrA2/Omi antibody that we obtained from a rabbit immunized against a mixture of two different human HtrA2/Omi peptides (amino acids 65–80 and 203–220) (1/1000), anti-AIF mAb (clone E1, Santa Cruz) (1/2000), anti-PARP mAb (clone C2-10, Pharmingen) (1/2000), anti-VDAC mAb (clone Ab4, Calbiochem) (1/6000), anti-Cox IV mAb (clone 10G8, Molecular Probe) (1/1000), anti-actin mAb (Sigma) (1/5000) and rabbit polyclonal antibodies specific for caspase-9 (Cayman Chemicals) (1/1000) or caspase-3 (Stressgen) (1/2000).

Techniques: Recombinant, Isolation, Incubation, SDS Page, Western Blot

Journal:

Article Title: Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization

doi: 10.1093/emboj/cdg423

Figure Lengend Snippet: Fig. 2. ΔΨm analysis in isolated mitochondria incubated with recombinant pro-apoptotic Bcl-2 members. Mitochondria isolated from HeLa cells were incubated with different concentrations (nM) of (A) recombinant oligomeric Bax or (B) recombinant tBid as in Figure 1. Then, ΔΨm was measured by flow cytometry using Rh123 (50 nM) as a probe. Incubation of isolated mitochondria with CCCP (10 µM) was used as a control. As in Figure 1, a fraction of mitochondrial pellets and supernatant fractions was also analyzed by western blotting. Blue arrows indicate a significant release of cytochrome c, Smac/Diablo and HtrA2/Omi (>50% release) and red arrows indicate complete or nearly complete release (>90% release).

Article Snippet: Antibodies used in immunoblotting were as follows: anti-cytochrome c mAb (clone 7H8.2C12, Pharmingen) (1/2000), rabbit polyclonal anti-Endo G (Pro-Sci Inc.) (1/1000), rabbit polyclonal anti-Smac/Diablo (Pro-Sci Inc.) (1/1000), a purified rabbit polyclonal anti-HtrA2/Omi antibody that we obtained from a rabbit immunized against a mixture of two different human HtrA2/Omi peptides (amino acids 65–80 and 203–220) (1/1000), anti-AIF mAb (clone E1, Santa Cruz) (1/2000), anti-PARP mAb (clone C2-10, Pharmingen) (1/2000), anti-VDAC mAb (clone Ab4, Calbiochem) (1/6000), anti-Cox IV mAb (clone 10G8, Molecular Probe) (1/1000), anti-actin mAb (Sigma) (1/5000) and rabbit polyclonal antibodies specific for caspase-9 (Cayman Chemicals) (1/1000) or caspase-3 (Stressgen) (1/2000).

Techniques: Isolation, Incubation, Recombinant, Flow Cytometry, Western Blot

Journal:

Article Title: Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization

doi: 10.1093/emboj/cdg423

Figure Lengend Snippet: Fig. 4. Cell-stress-associated EndoG and AIF release is prevented by zVAD-fmk, unlike cytochrome c, Smac/Diablo and HtrA2/Omi. (A) Percentages of cells with apoptotic nuclei (percentage of apoptotic nuclei) in HeLa cells treated for 7 and 9 h with staurosporine (STS, 2 µM) or actinomycin D (ActD, 20 µM) in the absence or presence of zVAD-fmk (100 µM). (B) Cells were treated as in (A). Total cell extracts were analyzed by western blotting for caspase-9 and caspase-3 processing and PARP cleavage. (C) HeLa cells were treated as in (A). Cytosolic fraction and heavy membrane fraction were analyzed by western blotting for the presence of cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF. As control for loading, actin was used in the cytosolic fraction and Cox IV in the heavy membrane fraction. (D) HeLa cells were treated as in (A) and then ΔΨm was assessed by flow cytometry using DiOC6 (50 nM). Top: % indicates percentage of ΔΨm loss. Bottom: histogram showing the percentage of ΔΨm loss in three independent experiments. The asterisks indicate additional non-specific bands.

Article Snippet: Antibodies used in immunoblotting were as follows: anti-cytochrome c mAb (clone 7H8.2C12, Pharmingen) (1/2000), rabbit polyclonal anti-Endo G (Pro-Sci Inc.) (1/1000), rabbit polyclonal anti-Smac/Diablo (Pro-Sci Inc.) (1/1000), a purified rabbit polyclonal anti-HtrA2/Omi antibody that we obtained from a rabbit immunized against a mixture of two different human HtrA2/Omi peptides (amino acids 65–80 and 203–220) (1/1000), anti-AIF mAb (clone E1, Santa Cruz) (1/2000), anti-PARP mAb (clone C2-10, Pharmingen) (1/2000), anti-VDAC mAb (clone Ab4, Calbiochem) (1/6000), anti-Cox IV mAb (clone 10G8, Molecular Probe) (1/1000), anti-actin mAb (Sigma) (1/5000) and rabbit polyclonal antibodies specific for caspase-9 (Cayman Chemicals) (1/1000) or caspase-3 (Stressgen) (1/2000).

Techniques: Western Blot, Flow Cytometry