Fig. 4

PDIA3P1 interacts with OCT4 in ESCC cells. A, B Online prediction of PDIA3P1 localization by lncLocator (A) and Locate-R (B). C Levels of PDIA3P1 from the nuclear and cytoplasmic fractions of KYSE-150 and Eca-109 cells were evaluated using qRT-PCR. β-actin and U6 were used as positive control for the cytoplasmic and nuclear fraction, respectively. D Online prediction of PDIA3P1 interacts with OCT4 by RPISeq. E Schematic representation of RNA-pulldown. F PDIA3P1 pull-down followed by Western blot validated its interaction with OCT4. G Schematic representation of RNA immunoprecipitation (RIP). H A RIP assay was performed using the OCT4 antibody in KYSE-150 and Eca-109 cells (***p < 0.001, student t-test), the data represent the mean ± S.D. of triplicates. I‐K The catRAPID was used to predict the interaction region between PDIA3P1 and OCT4. L Schematic diagrams of PDIA3P1 full-length and truncated fragments. M Truncated PDIA3P1 mapping of OCT4 binding domain. Top panel: RNA sizes of in vitro transcribed full-length and truncations of PDIA3P1. Bottom panel: Western blot of OCT4 pulled down by various PDIA3P1 fragments. N Prediction of PDIA3P1 structure was based on minimum free energy (MFE) and partition function. A red arrow indicates the OCT4-binding stem-loop structure. O The expression of OCT4 was detected by Western blot after transfected PDIA3P1 full-length or truncated fragments in KYSE-30 and KYSE-150 cells