Fig 2: ZEB1 coordinates other EMT-TFs in transcriptional regulation of EMT molecules. Transcriptional expression levels of (A) the epithelial marker E-cadherin (CDH1), (B) the mesenchymal marker N-cadherin (CDH2) detected by qPCR in NUM, UM, MetUM, OCM1 and C918 were compared. The presence of (C) CDH1 in the choroidal tissue but not in the tumor, and (D) more CDH2 in the tumor than in the choroid was detected by immunohistochemistry. (E) ZEB1 knockdown in both C918 and OCM1 and ZEB1 overexpression in OCM1 were validated by Western blots (WB). Knockdown of ZEB1 resulted in CDH1 accumulation in C918. Real-time qPCR analyses of the effects of (F) ZEB1 knockdown on the expression of the other EMT-TF genes including (G) ZEB2, (H) ID2, (I) SNAI1, (J) SNAI2, (K) TWIST1, and EMT marker genes including (L) CDH1, and (M) CDH2. (N) Chromatin immune-precipitated (ChIP) by ZEB1 antiserum (ZEB1) was utilized to detect the binding of ZEB1 to the EMT-TF genes SNAI1, TWIST1 and ID2, and to the EMT marker genes CDH1 and CDH2. Molecular weight (MW) marks were loaded on the far left lane for validation of ChIP-PCR amplicon size, followed by 10x diluted chromatin sample before immuneprecipitation (INPUT). Pan histone 3 (H3) and pre-immune serum (IgG) were served as a positive and a negative control, respectively, whereas any remaining chromatin after passing through the binding beads without any serum was served as a background control (Mock). Mean (M) ± standard deviation (SD). ‘*’ Indicates Student’s t-test p = 0.05, ‘**’ Indicates Student’s t-test p = 0.01.

Fig 3: Immunofluorescence of CDH2. Sections were obtained from either anterior (A, C) or posterior regions (B, D) in fixed bovine lenses (A, B) or fresh frozen bovine lenses (C, D). The sections were immunolabeled with anti- CDH2 antibodies. Images were obtained using either a Zeiss LSM 880 confocal laser scanning microscope (A, B) or a Zeiss Axioscan.Z1 (C, D). The results showed increased CDH2 signal at suture branches. (A, B) Scale bars, 200 µm. (C, D) Scale bars, 1 mm.

Fig 4: Overlay of connexin 50 (Cx50), cadherin (CDH2), and ARVCF signals. Overlay of MALDI images show inversely correlated expression of adherens junction proteins (N-cadherin [green] and ARVCF [red]) and complementary expression of gap junction protein connexin 50 [blue]). Scale bar, 1 mm.

Fig 5: MALDI images of CDH2 and ARVCF peptides. Images of three unique peptides from CDH2 and ARVCF normalized by root-mean-square (RMS). Increased abundance of CDH2 and ARVCF at the sutures was detected. Scale bar, 1 mm