Figure 3

Figure 3

(A–B) Progeny of papp-aa^+/-;Tg(igfbp5a:GFP) intercrosses were injected with BAC(igfbp5a:mCherry) containing the indicated gene. They were subjected to the low [Ca²⁺] stress test described in Figure 2A. Papp-aa-IRES-mCherry, Papp-aa E501A-IRES-mCherry, or mCherry expressing NaR cells were detected by GFP and mCherry expression. NaR cells expressing mCherry or Papp-aa-mCherry (yellow, double labeled by GFP and mCherry) were scored following a published scoring system (Liu et al., 2018). Representative images are shown in (A) and quantified data in (B). Scale bar = 50 µm. +, one cell division, -, no division. ****, p<0.0001 by Chi-square test. The total cell number is shown above the bar. (C) Conditioned media collected from HEK293 cells co-transfected with Igfbp5a and the indicated plasmid were analyzed by western blotting. Intact and cleaved Igfbp5a bands were indicated. (D–E) Tg(igfbp5a:GFP) fish were transferred to the low [Ca²⁺] medium containing 0–8 µM ZnCl₂ (D) or 200 µM Batimastat at three dpf (E). After two days of treatment, NaR cells were quantified and shown. n = 18 ~ 25 fish/group. **, p<0.001 by unpaired two-tailed t test. (F–H) Tg(igfbp5a:GFP) embryos were injected with BAC(igfbp5a:mCherry, BAC(igfbp5a:hSTC1-IRES-mCherry) (G) or BAC(igfbp5a:hSTC2-IRES-mCherry) (H). They were raised and subjected to the low [Ca²⁺] stress test described in Figure 2A. NaR cells expressing mCherry or human STC (yellow, double labeled by GFP and mCherry) were scored following a published scoring system (Liu et al., 2018). Representative images are shown in (F) and quantified results in (G and H). ++, two cell division, +, one cell division, -, no division during the experiment. ****, p<0.0001, Chi-square test. Total cell number is shown above the bar.

Figure 3—source data 1.