Effects of Röttlerin and Bay 11-7082 on the PMA-inhibited angiogenesis. (A) Treatment with PMA at 72 hours after post-fertilization significantly inhibited angiogenesis, and this inhibition was suppressed by co-treatment with Röttlerin (5 μM) or Bay 11-7082 (10 nM). Embryos were examined at 3 days post-fertilization. More than 20 embryos were examined in each experimental group. Twenty dechorionated Tg(fli1:EGFP)^y1 embryos were grown in a 6-well plate, and incubated in 2 mL solution containing PMA (20 ng/mL). Top panel: fluorescent images show defective vasculatures in ISV and DLAV at the PMA-treated Tg(fli1:EGFP)^y1 embryos. The arrowheads indicate the failure of forming the ISV or DLAV in the PMA-treated Tg(fli1:EGFP)^y1 embryos. Bar = 100 μm. Bottom panel: quantified results expressed by percentage of total ISV. (B) The expression of Thy-1 was detected in zebrafish embryos at 72 hours post-fertilization by Western blot analysis. The cropped blot was shown. The entire gel picture of 5B (Thy-1) was shown in the Supplemental Fig. S5. RÖ, Röttlerin.