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Fig. S2
(A) Acridine orange staining of stressed gata1:DsRed zebrafish indices cell death occurring in Gata1+ erythroid cells of the HCT. At 24 hpf, gata1:DsRed zebrafish were dechorionated and treated with 20 μg/5 mL 1-naphthol as before. At 72 hpf, the zebrafish were incubated in a solution of 1 μg/mL acridine orange (Sigma Chemical) dissolved in fish water for 40 minutes in the dark, and followed by three 5minute washed with fish water. The zebrafish were then anesthetized using Tricane (200 μg/mL) and embedded in 1% low melting agarose for imaging. Image acquisition to show the caudal hematopoietic tissue (CHT) region, was performed using a Leica DMI 6000 inverted epifluorescent microscope. Red, green, and composite fluorescence images were taken to identify Gata1+ erythroid cells (red) and overall cell death (green). Dying Gata1+ erythroid cells are shown in the overlay as yellow. Scale bar indicates 500 micros.
(B – F) At 24 hpf, gata1:DsRed zebrafish were dechorionated and treated with 20 μg/5 mL 1-naphthol as described in the Methods. At 72 hpf Gata1+ erythroid cells were sorted on a BD FACSAria II (BD Biosciences, San Jose, CA) into RNAlater (ThermoFisher Scientific, Waltham, Massachusetts) and RNA prepared according to the manufacture’s instructions. qRT-PCR of sorted cells was performed on a StepOne Plus thermocycler (Applied Biosystems, Waltham, Massachusetts ) using Taqman primer/probes sets (Dr03136839_m1 for sesn2, Dr03131895_m1 for nrf2 (nef2), Dr03434097_m1 for hmox1, Dr03146792_m1 for txn, and Dr03112085_m1 for tp53; all from ThermoFisher Scientific. n = 15 – 20 individual animals per group, technical duplicates, pooled from two independent experiments.
(G) 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. PFT was refreshed every 4 hours from 24 hpf to 72 hpf. ROS measured using the CellRox ROS probe relative to untreated group. One of three independent experiments. n = 20 – 30 individual animals per group showing one of two independent experiments.
(H) Flow cytometry histoplot of murine marrow cells stained with anti-CD71 (red) versus isotype control (blue). CD71 positive events were next assessed for ROS using CellRox Green and measuring mean fluorescence intensity. Blue histogram shows cells from a Tp53R270H/+ mouse without ROS induction. Green histogram shows CD71+ cells after treatment with 1 mM acrolein with increased ROS.
All data are shown as the mean ± SD, with the p-value from a Student’s t-test (unless otherwise indicated).