Fig 2: JAM-A does not regulate the HER2 transcriptional repressor YBX3/ZONAB in SK-BR-3 cells. (A,B) SKBR3 cells were transfected with control siRNA (siNEG) or a pool of JAM-A siRNA and harvested 72 h later for qRT-PCR (A) and immunoblot/densitometric (B) analysis of three independent experiments. Data represent mean ± s.e.m, compared using two-tailed, equal variance Student’s t-tests, *** p < 0.001. (C) SK-BR-3 breast cancer cells were transfected with 25 nM of control siRNA or a pool of JAM-A siRNA. Cells were harvested 72 h later and non-nuclear/nuclear fractions were prepared for Western blot analysis. The two bands for ZONAB reflect parallel detection of its long form and an alternately spliced shorter form. (D) SKBR3 cells were transfected with control siRNA or ZONAB Smartpool siRNA. After 72 h, RNA was extracted for RT-qPCR analysis. Experiments were performed three times and data represent mean ± s.e.m, compared using two-tailed, equal variance Student’s t-tests, *** p < 0.001.

Fig 3: The involvement of the ZO-1/ZONAB pathway in BK-induced rabbit corneal endothelial cell proliferation. (A) Representative western blotting gel images. Quantification analysis of (B) ZO-1, (C) nuclear ZONAB, (D) PCNA and (E) cyclin D1. Protein expression was normalized to ß-actin. Data are expressed as the mean ± standard deviation (n=5). *P<0.05, vs. control group; #P<0.05 vs. BK group; ?P<0.05 vs. NC-siRNA group. ZO-1, zonula occludens-1; ZONAB, ZO-1-associated nucleic-acid-binding protein; siRNA, small interfering RNA; BK, bradykinin; PCNA, proliferating cell nuclear antigen; NC, negative control.

Fig 4: BK upregulated the expression of tight junction ZO-1 and nuclear ZONAB in rabbit corneal endothelial cells. (A) Representative western blotting images for ZO-1 and ZONAB, with quantification of (B) ZO-1 protein expression and (C) nuclear ZONAB protein expressions. (D) Relative ZONAB mRNA levels, as determined by reverse transcription polymerase chain reaction. (E) The localization of ZONAB was evaluated using immunofluorescence. Nuclei were counterstained with DAPI. White arrows indicate ZONAB-positive cells stained green (magnification, ×400; scale bars, 20 µm). Data are expressed as the mean ± standard deviation (n=4). *P<0.05 vs. control group; **P<0.01 vs. control group. BK, bradykinin; ZO-1, zonula occludens-1; ZONAB, ZO-1-associated nucleic-acid-binding protein.

Fig 5: Knockdown of ZONAB expression in RCECs transfected with siRNAs. (A) Reverse transcription polymerase chain reaction and (B) western blotting analysis showed revealed that transfection of ZONAB siRNAs efficiently inhibited the expression of ZONAB mRNA and protein in RCECs, respectively. In particular, transfection with ZONAB-siRNA2 achieved an ~85% reduction of ZONAB mRNA. Data are expressed as the mean ± standard deviation (n=6). **P<0.01 vs. control group. ZONAB, ZO-1-associated nucleic-acid-binding protein; RCECs, rabbit corneal endothelial cells; siRNA, small interfering RNA.