Fig. 4

Genetic Inactivation of tp53 Leads to Elevated ROS, Cell Death, and Increased Mitochondrial Basal OCR

(A) Relative erythroid cell death indicated by propidium iodide staining cells after 1-naphthol exposure (20 μg/5 mL) and quantified by flow cytometry. n = 20–21 individual animals per group pooled from two independent experiments.

(B) Pro-oxidant exposure (0–30 μg/5 mL) leads to increased death and edema in tp53^M214K/M214K zebrafish (mutants denoted as tp53^m/m). Embryos were scored at 96 hpf (after 72 hr of pro-oxidant exposure). n ≥ 100/individual animals per group, p value from chi-square analysis. Data pooled from three independent experiments.

(C) Severe hemolytic edema in tp53^M214K/M214K zebrafish exposed to 1-napthol (20 μg/5 mL). Hemoglobin staining was performed using o-dianisidine.

(D) Heatmap of RNA-seq data from untreated and 1-naphthol-treated tp53^M214K/M214K zebrafish (treated from 24 to 72 hpf). Unsupervised clustering was performed on genes with log₂ expression >2-fold difference. n = 5 individual animals in each group.

(E) GO term analysis using the PANTHER database and software for GO enrichment analysis for biological process indicates genes involved in metabolism are highly upregulated in tp53^M214K/M214K after pro-oxidant challenge.

(F) Live image displaying erythroid precursors from gata1:DsRed × mito:GFP zebrafish at 72 hpf. Imaging performed with a Leica TCS SPE Spectral Confocal system. Scale bar represents 25 μm.

(G) Mitochondrial content of gata1:DsRed × mito:GFP and gata1:DsRed × mito:GFP × tp53^M214K zebrafish. Single-cell suspensions at 72 hpf were made and mitochondrial content determined by flow cytometry measuring GFP MFI. n = 18–20 individual animals per group in two independent experiments.

(H) Lack of OXPHOS reserve in tp53^M214/M214KK zebrafish. Mitochondrial OCR was measured using the Seahorse biochemical analyzer. Yellow bars indicate the amount of reserve OXPHOS, which is the difference between maximal OCR and basal OCR. Shown are the mean and SEM. n = 12–15 individual animals per group in two independent experiments. See also Figure S3D.

(I) Basal OCR is higher in tp53^M214K/M214K zebrafish. Shown are the means and SD. n = 12–15 per group showing one of two independent experiments.

(J) Quantification of OXPHOS reserve indicates tp53^M214K/M214K zebrafish have no reserve. Shown are the means and SD. n = 12–15 per group showing one of two independent experiments. All pro-oxidant exposure times were from 24 to 72 hpf, except (C) and (I and J), which were from 24 to 96 hpf and from 24 to 120 hpf, respectively.

(K) Pro-oxidant exposure (20 μg/5 mL) combined with oligomycin exposure leads to decreased death and edema in tp53^M214K/M214K zebrafish. Embryos scored at 96 hpf after 72 hr of drug exposure. n ≥ 100/individual animals per group, p value from chi-square analysis. Data pooled from three independent experiments.