Fig. 3: Identification and quantitation of endogenous authentic OCT4A proteins in somatic cancer cells.

a Generation and verification of CRISPR/Cas9-mediated OCT4A-specific knockout (KO) cancer cell models. Upper panel: the strategy to obtain OCT4A-KO cancer cell clones. Note both single gRNA (gRNA2) and dual gRNAs (gRNA1 + gRNA2) were used to generate the OCT4A-KO HeLa cell clones. Lower panels: verification of homozygous OCT4A-KO clones by PCR (Left, A2 clone) or PCR-RFLP (Middle and Right, 1-1 and 2-2 clones). b Enhanced WB analysis of NF and CF of wild-type HeLa (WT) and OCT4A-KO HeLa clones (2-2, 1-1 and A2) with anti-OCT4A (CST 2890). c WT and OCT4A-KO clones subjected to Casilio activation followed by regular WB analysis. d−f Generation and characterization of CRISPR/Cas9-mediated knockin (KI) Tag-OCT4A HeLa cell clones. The strategy of gene KI to obtain Tag-OCT4A HeLa clones mediated by CRISPR/Cas9 and homologous recombination (d). Immunostaining of a single allele tagged Tag-OCT4A HeLa clone (3A11) with anti-OCT4A and anti-FLAG. Scale bars, 20 μm (e). IP and LC/MS/MS analysis of 3A11 HeLa clone for OCT4A identification (f). g Quantitation of endogenous OCT4A proteins in HeLa and NCCIT using enhanced WB analysis with recombinant His-OCT4A as a standard. Upper panel: enhanced WB analysis of His-OCT4A, NF of NCCIT (NT1: 1:20,000 dilution and NT2: 1:40,000 dilution) and HeLa cells, with NF of A2 clone as negative control. Middle panel: Standard curve of molar concentration and band intensity (gray value) for His-OCT4A. Lower panel: The calculated OCT4A protein molecules per cell nucleus based on absolute His-OCT4A protein concentrations determined by the above standard curve, and normalization by sample dilution factors and harvested cell numbers