Fig. 5
a. Experimental design for the investigation of developmental consequences of TRIM21-mediated protein depletion in zygotes. b. Representative images of blastocysts developed in KSOM (aa) after the microinjection of mCherry-Trim21 mRNA and OGDB tracer with or without TEAD4 antibody. Effect of anti-TEAD4 was due to TRIM21-mediated depletion, as shown by the lack-of effect of TEAD4-antibody alone. c. Representative images of embryos with mCherry-Trim21 mRNA and OGDB tracer; embryos with mCherry-Trim21 mRNA, OGDB tracer and anti-TEAD4; non-injected oocytes with no fluorescence. Plot shows Cherry fluorescence intensities of zygotes and subsequent stages preloaded with mCherry-Trim21 mRNA and OGDB tracer, and then injected with water (
), anti-GFP antibody (
) or anti-TEAD4 antibody (
), compared to the background fluorescence of non-manipulated cells (
). N = 3 zygotes or embryos per stage per treatment. Note the secondary right axis used in plot to better discern the background fluorescence values. d. Representative immunofluorescent signals (largest cross section, nucleus fluorescence) of TEAD4 and CDX2 in TRIM21-only and TEAD4-depleted embryos at day E3.5 (n = 7 TEAD4-depleted and n = 8 TRIM21-only embryos for TEAD4 immunofluorescence; n = 11 TEAD-depleted and n = 8 TRIM21-only embryos for CDX2 immunofluorescence). DNA stained with YO-PRO-1. Arrows point at peripheral nuclei that are TEAD4- or CDX2-positive in controls but negative in TEAD4-depleted embryos. Size bar, 50 μm. OGDB, Oregon Green dextran beads. Error bars = standard deviations. Statistical significance tested with Student’s t test. AU, arbitrary units of fluorescence intensity