Fig. 3

Oxidative Stress Induces tp53, which Can Modulate ROS Levels

(A) Zebrafish show increased 8-oxo-dG after pro-oxidant exposure (1-naphthol at 20 μg/5 mL). n = 5–6 per condition, one of two independent experiments.

(B) Representative image of a TUNEL assay from zebrafish after pro-oxidant exposure (20 μg/5 mL). Boxed region indicates the CHT. Scale bar represents 500 μm.

(C) Number of TUNEL-positive cells present in the CHT. n = 14–16 individual animals per condition in one of two independent experiments. ^∗p < 0.0001.

(D) Heatmap of RNA-seq data from untreated and 1-naphthol-treated zebrafish. Unsupervised clustering was performed on genes with log₂ expression >2-fold difference.

(E) Distribution of genes with log₂ expression >2-fold difference between control and 1-naphthol-treated zebrafish. n = 5 control animals and n = 6 naphthol-treated animals.

(F) IPA results of the significant Upstream Regulators having greater expression in 1-naphthol-treated zebrafish.

(G) RT-PCR of tp53 in zebrafish after pro-oxidant exposure (n = 20 individual animals per group, technical duplicates, pooled from four independent experiments). ^∗p < 0.05, ^∗∗p < 0.01.

(H) Tp53 inactivation using pifithrin (PFT) during pro-oxidant exposure (1-naphthol at 30 μg/5 mL) at 1 μM final concentration increases ROS in Gata1⁺-expressing erythrocytes. ROS measured using the CellRox ROS probe relative to untreated group. n = 20–25 individual animals per condition showing one of three independent experiments. See also Figure S2G.

(I) Elevated ROS in tp53^M214K zebrafish Gata1⁺-expressing erythroid cells after pro-oxidant challenge. n = 20–25 individual animals per condition showing one of three independent experiments.

(J) Erythroid progenitors in TP53^R270H/+murine whole bone marrow have increased ROS generation after short-term oxidative challenge. After exposure to 1 mM acrolein, nucleated CD71⁺ cells were gated by flow cytometry (see Figure S2H for gating) and ROS measured using CellROX green probe. ROS is given relative to the untreated group. Shown are data pooled from two independent experiments. All zebrafish pro-oxidant exposure times were from 24 to 72 hpf. n = 10–20 individual animals per condition pooled from two independent experiments.

All data are shown as the mean ± SD, with the p value from a Student t test.