Fig. 8

Visualization and quantification of the amount of apoptosis after MO-based knockdown (without and with mRNA rescue), CRISPR/Cas9-knockout of TPC2, or following pharmacological treatment with bafilomycin A1 or trans-ned-19. Embryos were (A-C) untreated or (D-I) injected with: (D) ~5 ng standard control-MO; (E) ~7.5 ng p53-MO; (F) ~2.5 ng TPCN2-MO-T with p53-MO; (G) ~5 ng TPCN2-MO-S with p53-MO; (H) ~1.3 ng TPCN2-MO-T+~2.5 ng TPCN2-MO-S with p53-MO; or (I) ~2.5 ng TPCN2-MO-T with p53-MO and ~50 pg tpcn2 mRNA. (J,K) Representative (J) heterozygous and (K) homozygous tpcn2 mutants are also shown. In addition, wild-type embryos were treated with: (L) 1% DMSO; (M) bafilomycin A1 at 5 µM; or (N) trans-ned-19 at 500 µM. (A-N) All the embryos were then fixed at ~24 hpf, and cells undergoing apoptosis were labeled using the TUNEL assay. These are bright-field images onto which are superimposed the respective fluorescence images showing TUNEL-positive cells in red. The yellow rectangles show the size and location of the regions of interest (ROIs) used for quantification. (A,B) Representative TUNEL (A) positive and (B) negative controls. In panels (L-N), the black arrowheads indicate the elevated levels of apoptosis in the region of the trunk damaged during tail excision. (O) Bar chart to show the mean ±SEM fluorescence intensity within the ROIs (n=3). Statistical analysis was carried out using one-way ANOVA. The asterisk indicates the significance difference (at p<0.001) between the TUNEL positive control and the other groups, as determined using the Tukey's post hoc method. No other significant differences were found among the groups. Scale bar, 25 µm.