Fig. 2

PDIA3P1 upregulates OCT4 to promote the cancer stem cell properties in ESCC cells. A, B Representative images of spheres and histogram analysis of sphere-formation rates in the indicated cells. Silencing PDIA3P1 in TE-1 and Eca-109 cells (A) and overexpression of PDIA3P1 in KYSE-30 and KYSE-150 cells (B) (**p < 0.01, student t-test). C, D qRT-PCR shows expression levels of esophageal CSCs cell surface markers after transfection with PDIA3P1 siRNA (C) and expression plasmid (D) (**p < 0.01, ***p < 0.001, student t-test). E Flow cytometry analysis of percentage of SP cells after PDIA3P1 knockdown (**p < 0.01, student t-test). F The percentage of SP cells was detected by flow cytometry after overexpression of PDIA3P1 (**p < 0.01, student t-test). G, H Flow cytometry analysis percentage of CD271 + /CD44 + cells, (G) PDIA3P1 was knocked down in TE-1 and Eca-109 cells, and overexpression of PDIA3P1 in KYSE-30 and KYSE-150 cells (H) (***p < 0.001, student t-test). I, J Expression levels of several key components of drivers of cancer stem cell properties in cells were analyzed by western blotting. I Silencing of PDIA3P1. J Over-expression of PDIA3P1. K Representative images of immunofluorescence staining revealing the effect of PDIA3P1 knockdown on the expression of OCT4 in Eca-109. L Representative images of immunofluorescence staining revealing the effect of PDIA3P1 overexpressed on the expression of OCT4 in KYSE-150. M Western blotting shows the levels of OCT4 in normal cell line HEEC and five ESCC cell lines (KYSE-30, KYSE-150, KYSE-520, TE-1 and Eca-109). N OCT4 expression detected in 6 paired ESCC tissues and non-tumor specimens by western blotting. O Eca-109 and KYSE-150 cell lines were transfected with siOCT4, the expression of OCT4 was analyzed by western blotting. P-R KYSE-150 cells were transfected with a control, or PDIA3P1 expression plasmid or the combination of PDIA3P1 expression plasmid and OCT4 siRNA. After 48 h analyzed CSCs properties. P Sphere-formation. Q Percentage of SP cells. R Percentage of CD271 + /CD44 + cells (**p < 0.01, ***p < 0.001, for difference from the transfected with vector and si-NC cells by ANOVA with Dunnett's correction for multiple comparisons), All, the data represent the mean ± S.D. of triplicates