Fig. 3

Knockdown of beclin 1 suppresses abnormal autolysosomal puncta formation and embryonic senescence caused by Spns1 deficiency in zebrafish.

(A) Effect of beclin 1 knockdown on EGFP-LC3 puncta formation in spns1-depleted zebrafish embryos. Injection of control (water) injection, spns1 MO (4 ng/embryo) or coinjection of spns1 MO (4 ng/embryo) and beclin 1 MO (12 ng/embryo) into Tg(CMV:EGFP-LC3) fish was performed to assess whether the beclin 1 knockdown reduces or eliminates aggregated LC3 puncta induced by Spns1 depletion at 84 hpf. Scale bar, 10 μm. Quantification of data presented in panel A (n = 12) is shown in the right graph; the number (n) of animals is for each morphant or water-injected control. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (B) Effect of beclin 1 knockdown on EGFP-GABARAP as well as mCherry-LC3 puncta formation in spns1-depleted zebrafish embryos. Injection of control (water), spns1 MO or coinjection of spns1 MO and beclin 1 MO into Tg(CMV:EGFP-GABARAP;mCherry-LC3) fish was performed to evaluate whether the beclin 1 knockdown reduces or eliminates the aggregation of GFP-GABARAP puncta in comparison with those of LC3 caused by the Spns1 depletion at 84 hpf. Scale bar, 10 μm. Quantification of data presented in the top row (green; EGFP) (n = 9), middle row (red; mCherry) (n = 12), and bottom row (yellow; merge of EGFP and mCherry) (n = 9) in panel B is shown in the right graphs; the number (n) of animals is for each morphant or water-injected control. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (C) Effect of beclin 1 knockdown on embryonic senescence in spns1 morphant. By using the same injection samples [injection of control (water), spns1 MO or coinjection of spns1 MO and beclin 1 MO into Tg(CMV:EGFP-GABARAP;mCherry-LC3) fish], SA-β-gal staining was performed to assess whether the beclin 1 knockdown has any impact on the embryonic senescence caused by Spns1 depletion at 84 hpf. Representative images of individual fish by bright field (BF, live samples) and SA-β-gal (SABG) staining are shown in the upper and middle panels, respectively. Scale bar, 250 μm. Lower panels are larger magnification images of corresponding SA-β-gal samples shown in the middle panels and the fluorescence images of nuclei counterstained with DAPI. Scale bar, 10 μm. Quantification of data presented in the middle row (SABG) in panel C (n = 12) is shown in the right graph; the number (n) of animals is for each morphant or water-injected control. Error bars represent the mean ± S.D., *p<0.005.