Ab151733

DNA and associated proteins on chromatin in cultured cells were crosslinked by 1% formaldehyde for 15 min at 37 °C. Cells were then scraped and collected in cellular lysis buffer (5 mM Pipes, 85 mM KCl, 0.5% NP-40, protease inhibitors). Cytoplasmic lysates were discarded and nuclear components were resuspended in nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 0.2% SDS, protease inhibitors) and sonicated for 10 min (Covaris). Approximately 4 μg of KLF4 antibody (Abcam ab151733) or control Rabbit IgG were incubated with 25 μl of protein G magnetic beads for 6 h at 4 °C, and then incubated with 100 μg of cleared chromatin overnight at 4 °C. After three washes, immunoprecipitated material was eluted at 55 °C for 1 h with 10 μg ml⁻¹ proteinase K, and then decrosslinked at 65 °C for 4 h (ref. 42 (link)). The primer sequences used for ChIP-qPCR are listed in Supplementary Table 7.

COMMD1 (NBP2-4633, Novusbio), ZEB1 (NBP2-13159, Novusbio), Sox2 (ab97959, Abcam), KLF4 (ab151733, Abcam), Oct-4 (2750, Cell Signaling), MYC (5605, Cell Signaling), NANOG (D73G4)(4903, Cell Signaling), K8/18 (20R-Cp004, Fitzgerald Industries), P53 (SC-126, Santa Cruz Biotechnology), Phospho-NFκB p65 (Ser536) (93H1)(3033, Cell Signaling), HIF1A(610959, BD), HIF2A(NB100-122, Novusbio), MCT4(sc-50329, scbt), CAV1(610407, BD), B-actin (A5441, Sigma), tubulin (T4026, Sigma), FITC-conjugated anti-CD24, PE-conjugated anti-CD24, APC-conjugated anti-CD44, APC-conjugated anti-PD-L1 (BD Biosciences).

iPSCs were fixed in 4% paraformaldehyde and stained with the following antibodies: Oct4 (1/100 dilution, SC-9081, Santa Cruz, CA, USA), KLF4 (1/250 dilution, Ab151733, Abcam, Cambridge, UK), Sox2 (1/100 dilution, 630801, BioLegend, San Diego, CA, USA), TRA-1-60 (1/200 dilution, MAB4360, Millipore, Billerica, Massachusetts, USA), TRA-1-81 (1/100 dilution, MAB4381, Millipore), and SSEA-4 (1/200 dilution, MAB4304, Millipore). Alexa Fluor 594- (1/400 dilution, A11037, Life Technologies) and 488-conjugated secondary antibodies (1/400 dilution, A11029, Life Technologies) were used and cells were detected by immunofluorescence microscopy.