Fig. 4

Ectopic expression of Bcl2 inhibits excessive autophagic flux in kri1l^cas002 HSPCs. (A) Representative immunoblotting images of Lc3-I, Lc3-II, Beclin1 and p62 in whole embryo lysates of wild-type (WT) and kri1l^cas002 embryos at 3 dpf, treated with or without chloroquine (5 μM) for 6 h before harvest. Tubulin serves as the loading control. (B) Statistics of the average number of autophagosome (indicated by mCherry-Lc3 puncta) in kri1l^cas002 mutant embryos and WT siblings with or without chloroquine treatment at 3 dpf. mCherry-Lc3 puncta are counted in over 20 EGFP+ cells (in cmyb:egfp background) per embryo, total 6-8 embryos under each condition. Without chloroquine treatment, P = 0.0040; with chloroquine treatment, P = 0.0008. Error bars represent SEM. **P ≤ 0.01; ***P ≤ 0.001 (Student’s t-test). (C-N) Representative confocal images of mCherry-Lc3 puncta (autophagosomes) in HSPCs of live zebrafish embryos. Wild type siblings or kri1l^cas002 mutant embryos in the Tg (cmyb: egfp) transgenic background, were injected with mcherry-lc3 mRNA at one-cell stage and imaged at 3 dpf with or without pre-treatment with chloroquine (5 μM) for 6 h. Scale bars: 5 μm. (O) Immunoblotting analysis of whole embryo lysates from WT andkri1l^cas002 mutant embryos with or without bcl2 mRNA injection. (P) Statistics of the average number of autophagosome for kri1l^cas002 mutant embryos and wild type siblings with Bcl2 overexpression at 3 dpf. mCherry-Lc3 puncta are counted in over 20 EGFP+ (cmyb: egfp) cells per embryo, total 8-11 individual embryos. P = 0.0021. Error bars represent SEM. **P ≤ 0.01; ***P ≤ 0.001 (Student’s t-test). (Q-S) Representative confocal images of mCherry-Lc3 puncta (autophagosomes) in HSPCs of live zebrafish embryos with Bcl2 overexpression. Scale bars, 5 μm.