Ab171380

The GSCs were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors for 30 min at 4 °C. Then the supernatants were collected after centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatants were mixed with loading buffer (Boster, Wuhan, China), and equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred to PVDF membranes. The PVDF membranes were blocked and incubated with the primary antibodies at 4 °C overnight. The primary antibodies were used at the following dilutions: rabbit anti-β-actin (1:2000; #4970, CST), mouse anti-β-III tubulin (1:1000; #4466, CST), rabbit anti-GFAP (1:1000; BA0056, Boster), mouse anti-sox2 (1:1000; ab171380, Abcam), rabbit anti-CD133 (1:1000; ab19898, Abcam), rabbit anti-H3 (1:1000; #4499, CST), rabbit anti-OCT4 (1:1000; ab18976, Abcam), rabbit anti-acetyl-H3 (1:1000; #06-599, Millipore), rabbit anti-H3K27me3 (1:1000; #9733, CST), mouse anti-H3K9me3 (1:1000; #5327, CST), rabbit anti-EZH2 (1:1000; #5246, CST). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies at room temperature. The antibody labeling was detected using an enhanced chemiluminescence reagent (Merck Millipore). The protein bands were analyzed using ImageJ.

The frozen primary or PDX breast cancer tissues were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, blocked with 3% BSA, and stained with an anti-TRIB3 (1:100, Abcam, ab137526), anti-CD68 (1:100, Abcam, ab955), anti-FOXO1 (1:100, Abcam, ab52857), or anti-SOX2 (1:100, Abcam, ab171380) primary antibody followed by Alexa Fluro 488 and/or Alexa Fluro594 secondary antibodies (1:200, Life Technologies). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained by using a laser scanning confocal imaging system (Olympus Microsystems).

Immunohistochemical staining was performed using Dako REALTM EnVision^TM Detection System (K5007, Dako, Glostrup, Denmark) in accordance with the manufacturer’s protocol. Rabbit anti-human VDAC2 polyclonal antibody (ab126120, Abcam) and mouse anti-human SOX2 (ab171380, Abcam) were used as the primary antibodies. Immunohistochemical scores of VDAC2 and SOX2 were calculated independently by two histopathologists in a blinded manner according to the staining intensity and percentage of positive tumor cells^{68 (link)}. The best predictive cutoff value of VDAC2 expression was determined to be 6 analyzed by X-tile software. The score ≥ 6 was defined as VDAC2^high, otherwise was defined as VDAC2^low.

Fixed and stored embryos were rehydrated with 75%, 50%, 25% MeOH in PBST and washed several times with PBST. Embryos were digested with Proteinase K (10 µg/ml) in PBST for 45 min at RT. Embryos were re-fixed with 4% PFA for 20 min at RT and then washed several times with PBST. Larvae were blocked in blocking solution [1% bovine serum albumin (BSA), 5% goat serum, 1% DMSO in PBST] for 1 h and incubated with primary antibodies diluted in blocking solution. The anti-Sox2 antibody (20G5, Abcam, ab171380, LOT GR3253929-5; cross-reacts with both zebrafish Sox2 and Sox3 proteins; Westphal et al., 2022 (link)) and the anti-phospho-Histone H3 (Ser10) antibody (Merck, 06-570, LOT 3319395) were diluted 1:400. Embryos were incubated in primary antibodies overnight at 4°C and then washed several times for 30 min each with PBST supplemented with 1% DMSO. Embryos were incubated with the secondary antibodies 1:1000 in PBST supplemented with 1% DMSO and 1% blocking reagent (Roche, 1096176) overnight at 4°C. Secondary antibodies were Alexa 555 goat anti-mouse IgG (Thermo Fisher Scientific, A-11001) and Alexa 488 goat anti-rabbit IgG (Thermo Fisher Scientific, A-11070). Embryos were washed several times in PBST, transferred to 80% glycerol in PBST and stored at 4°C protected from light. The embryos were recorded soon after staining.

These experiments were performed as described previously^{21 (link),23 (link),54 (link)–57 (link),59 (link)}. Mice were anesthetized and perfused with phosphate-buffered saline followed by 4% paraformaldehyde (PFA). Brain tissues were then dissected and fixed in 4% PFA overnight at 4 °C. Fixed brain tissues were processed for paraffin embedding and then cut into 5-μm sections. Methods used for immunofluorescent staining have been previously described^{60 (link)}. Primary antibodies used for immunofluorescent staining were anti-REST (HPA006079, Sigma), anti-Drd2 (sc-5303, Santa Cruz Biotechnology), anti-Sox2 (ab171380, Abcam), and anti-Nestin (ab6142, Abcam). TUNEL assay was performed using a TUNEL apoptosis kit purchased from Roche (11684817910). Secondary antibodies used in immunofluorescent staining were conjugated with either Alexa 488 or Alexa 555 (Life Science Technologies). Staining results were viewed and photographed using confocal microscopy.