Fig 2: CHAF1A and PCNA facilitate DNA replication in EC cells. (A,C) Flow cytometry analysis of the cell cycle (left panel) and (B,D) S–phase quantification (n = 3; right) in KYSE30 cells transfected with siNC, siPCNA, or siCHAF1A. (E) Schematic of alternative 5–chloro–2–deoxyuridine (CldU)/5–iodo–2–deoxyuridine (IdU) pulse–labeling protocol to evaluate fork length in KYSE30 cells transfected with siNC, siPCNA, or siCHAF1A. (F) Representative images and scatterplots of IdU tract length for individual forks (n = 100). Flow cytometry analysis of the cell cycle was performed using ModFit software. * p < 0.05, ** p < 0.01, n.s.: non–significance.

Fig 3: CHAF1A and PCNA knockdown reduce EC cell proliferation. (A,C,E,G) Western blot analysis of PCNA or CHAF1A knockdown in KYSE30 and KYSE510 cell lines. (B,D) Representative images (left) and colony quantification (n = 4; right) of KYSE30 cells (B) and KYSE510 cells (D) with PCNA knockdown. (F,H) Representative images (left) and colony quantification (n = 4; right) of KYSE30 cells (F) and KYSE510 cells (H) with CHAF1A knockdown. ** p < 0.01.

Fig 4: Double knockdown of CHAF1A and PCNA increases the proportion of EC cells in S–phase. (A,B) Flow cytometry analysis of EdU and propidium iodide (PI) staining in KYSE30 cells transfected with siNC, siPCNA, or siCHAF1A. The percentage of S phase cells is shown (n = 3). (C) Knockdown efficiency was detected using western blotting. (D,E) Schematic of cell cycle synchronization in KYSE30 transfected with siNC, siPCNA, or siCHAF1A. Flow cytometry analysis of the cell cycle was performed using FlowJo software. (F,G) Representative images (left) and colony quantification (n = 3; right) of KYSE30 cells. siRNA pools were used for single or/and double PCNA and CHAF1A knockdown. * p < 0.05, ** p < 0.01.

Fig 5: Expression, localization, and interaction of CHAF1A and PCNA in EC cells. (A) CHAF1A and PCNA expression in 17 EC cell lines detected by western blotting. (B) Cytoplasmic (Cyto), soluble nuclear, and chromatin distribution of PCNA and CHAF1A in KYSE30, KYSE450, and KYSE510 cells subjected to cell fractionation assays and western blotting. Lamin B1 was used as a chromatin marker, and GAPDH was used as a cytoplasmic marker. (C) Co-localization of PCNA-CHAF1A was determined using a PLA probe in KYSE30 cells. Scale bars, 5 µm.