Journal: bioRxiv
Article Title: Disruption of ADAMTSL4 Causes Ectopia Pupillae in Zebrafish via COL8A1-Driven Cell Migration
doi: 10.1101/2025.11.03.686242
Figure Lengend Snippet: (A). qPCR analysis showing fold changes in mRNA expression of ECM-associated genes in EP eyes of adamtsl4 ⁻/⁻ zebrafish mutants compared with normal eyes of sibling controls. Each group included three biological replicates. (B). qPCR validation of ECM-associated gene expression in human RPEs following siRNA-mediated knockdown of ADAMTSL4 , compared to negative siRNA controls. Each group has three biological replications. (C). Western blot analysis of ADAMTSL4, TGFB2, and COL8A1 protein levels in ADAMTSL4 knockdown RPEs versus controls. GAPDH was used as a loading control. Each group contained three biological replicates. (D). Co-IP between ADAMTSL4 and COL8A1 in HEK293T cells. Exogenous ADAMTSL4 fused with EGFP and COL8A1 fused with mCherry were expressed and precipitated using EGFP antibody-coated beads. Interacting proteins were detected by Western blot. (E). Knockdown of COL8A1 in ADAMTSL4- deficient RPE cells using siRNA. Knockdown efficiency was confirmed by qPCR. (F). Wound healing assay showing significantly reduced cell migration in RPEs following ADAMTSL4 knockdown, COL8A1 knockdown, combined knockdown. Negative siRNA control was transfected toaccount for potential cytotoxic effects of transfection, and total siRNA concentrations were maintained consistently across all groups. Quantification was based on the percentage change in wound area across at least three independent experiments. Statistical significance is indicated by asterisks: ns P ≥ 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; error bars indicate the standard error of the mean. Co-IP, Co-Immunoprecipitation; qPCR, quantitative polymerase chain reaction; siRNA, small interfering RNA; RPE, retinal pigmented epithelium; HEK293T, Human Embryonic Kidney 293T cell; IP, immunoprecipitation; IB, immunoblotting.
Article Snippet: The primary antibodies used for immunoblotting were as follows: ADAMTSL4 Rabbit pAb (Abclonal, #A4785, 1:1000), TGFB2 Rabbit pAb (Abclonal, #A3640, 1:1000 dilution), Collagen VIII alpha 1 Rabbit pAb (Affinity, #DF8902,1:1000 dilution), GFP tag Mouse mAb (Proteintech, #66002-1-lg, 1:20000 dilution), mCherry Rabbit pAb (Proteintech, #26765-1-AP, 1:2000), and GAPDH Rabbit pAb (Proteintech, #10494-1-AP, 1:5000 dilution).
Techniques: Expressing, Biomarker Discovery, Gene Expression, Knockdown, Western Blot, Control, Co-Immunoprecipitation Assay, Wound Healing Assay, Migration, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Small Interfering RNA
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