Ab18976

Colorectal cancer stem cells were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) with protease inhibitors. The lysates were separated on SDS-polyacrylamide gels and transferred to 0.22 μm PVDF membranes. The membranes were incubated with 5% skim milk for 2 h at RT and incubated with the primary antibodies at 4 °C overnight. Primary antibodies against the following molecules were used at the following dilutions: PCGF1 (1:1000; ab183499, Abcam), CD133 (1:1000; ab19898, Abcam), SOX2 (1:1000; ab97959, Abcam), Oct4 (1:1000; ab18976, Abcam), cleaved PARP1 (1:1000; ab32064, Abcam), H3K4me3 (1:1000; CST#9751, CST), H3K27me3 (1:1000; CST#9733, CST), H3K9me3 (1:1000; CST#13969, CST), H3K9/K14ac (1:1000; CST#9677, CST), H3K18ac (1:1000; CST#9675P, CST), H3 (1:1000; CST#4499, CST), and β-actin (1:1000; HC201, TransGen Biotech). The membranes were incubated with HRP-conjugated goat anti-mouse (1:5000; HS201-01, TransGen Biotech) or goat anti-rabbit (1:5000; HS101-01, TransGen Biotech) secondary antibodies for 1 h at room temperature after washing. Protein expression was detected with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). The protein bands were analysed using ImageJ software (64-bit).

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.

Cultures of Pax6-GFP mES cells were fixed in 4% paraformaldehyde (PFA)/4% sucrose and processed for immunostaining. Commercial antibodies for Oct4 (ab18976; Abcam, Cambridge, MA), SSEA1 (FCMAB117P; Millipore, Billerica, MA) and Nanog (ab106465; Abcam, Cambridge, MA), and histochemical reagents for alkaline phosphatase activity (Sigma-Aldrich, St. Louis, MO) were used for marker studies. Differentiation of Pax6-GFP mES cells was evaluated by immunostaining with antibodies to neurofilament (NF, ectoderm) (ab24575; Abcam, Cambridge, MA), alpha-fetoprotein (AFP, endoderm) (sc-8108; Santa Cruz Biotech.) and smooth muscle actin (SMA, mesoderm) (ab5694; Abcam, Cambridge, MA). Primary and secondary antibody immunostaining were performed as previously described [38] (link). Controls included omission of primary or secondary antibody, and comparison of differentiated and undifferentiated cells. For antibody specifications, see S1 Table.

The same three anti-OCT4 antibodies were used for ICC: sc-5279 (Santa Cruz Biotechnology), ab19857, and ab18976 (both from Abcam). The following secondary antibodies were used: anti-mouse AlexaFluor568 and anti-rabbit AlexaFluor568. AlexaFluor488-conjugated phalloidin (Invitrogen) was used to stain actin filaments and DAPI to stain DNA. Images were obtained with appropriate excitation/emission on Leica confocal microscope using identical image acquisition settings.

MDA-MB-231 seeded onto coverslips or homing on TAH for 24h were fixed by 4% paraformaldehyde for 1.5 h. Blocking was carried out with BSA-PBST (BSA: 1%, PBST: phosphate buffered saline with 0.01% tween 20, v/v) at room temperature for 1 h. PBST was used for washing and in the dilution of anti-Oct4 (1:200; ab18976, Abcam), anti-Sox2 (1:200; D1C7J, Cell Signaling), Alexa Fluor 488, and Alexa Fluor 594-conjugated donkey anti rabbit secondary antibodies (both 1:200; R37118 and R37119, Thermo Fisher Scientific). All antibody-antigen reactions were carried out at room temperature for at least 1 h. DAPI was diluted as 1:1000 in PBST for nucleus staining at room temperature for 15 min after removing excess secondary antibodies with PBST washing. Immunofluorescence images were obtained as described in Section 4.5.

For this study, we used anti-OCT4 (1:200 for IF, 1:500 for WB, ab18976 Abcam), anti- SOX2 (1:500 for IF, 1:1000 for WB, ab97959 Abcam), anti-NANOG (1:50 for IF, 1:500 for WB, OAAB11202, Aviva Systems Biology), anti-CDX2 (1:100 for IF, ab15258 Abcam; 1:500 for WB, ab88129 Abcam), anti-beta actin (1:1000 for WB, ab8227 Abcam) and Anti-LASP1 (1:1000 for WB, ab156872 Abcam).