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Fig. 5
Knockdown of EPO and EPOR increased apoptosis within zebrafish pronephros. A. Increased apoptotic cell number (white arrows) and altered pronephros structure in EPO MO. and EPOR MO. as compared to Control MO. Confocal images of TUNEL-stained TG(WT1B:EGFP) zebrafish embryos at 48 hpf are shown. The yellow square highlighted glomerulus of zebrafish pronephros. White arrows labeled apoptotic cells shown in red color; pronephros was labeled in green. White scale bar: 100 μm. B. Quantification of glomerulus and total pronephros apoptotic cells (TUNEL) in EPO MO., EPOR MO. and Control MO. performed in three independent experiments as shown in Figure A (n = 28–32 embryos per group). C. Quantification of glomerulus and total pronephros apoptotic cells (TUNEL) in EPO CRISPRants, EPOR CRISPRants, and Control CRISPRants shown in Supplementary Figures performed in three independent experiments. (n = 28–30 embryos per group). D. FACS scan results for Annexin V stain in EPO MO. and EPOR MO. as compared to Control MO. E. Increased relative Annexin V-positive/7-AAD-negative cell fraction as an indicator of early apoptotic cells in EPO MO. and EPOR MO. as compared to Control MO. performed in at least three different experiments. Cells from 48 hpf zebrafish were harvested, stained with Annexin V-APC and 7-AAD and analyzed by FACS. F. Increased relative Annexin V-positive/7-AAD-positive cell fraction indicating late apoptotic cells in EPO MO. and EPOR MO. as compared to Control MO. performed in at least three different experiments. Data were analyzed using the Student‘s t-test (C and D). Mean ± s.e.m. ns not significant. ***p < 0.001.