RNA-seq of erythroid gene expression after 7,17-diHDPAn-3 treatment. (A) Heat map showing the expression profiles of 12 differentially expressed genes (P adj < .05; |log₂ fold change| ≥ 1) in FACS-sorted GATA1-positive erythroid cells from control and 7,17-diHDPAn-3–treated zebrafish embryos. RNA-seq was performed using 4 biological replicates per group, with 200 000 sorted cells per sample. (B) Volcano plot highlighting significantly upregulated (fh, klhl43, hsp90aa1.1, stab2, klhk41b, mrc1a, unc45b, and gpm6bb in red) and downregulated (xpo1b, carmil2, atf7ip, and si:ch211-195b15.8 in green) genes in treated samples vs controls. (C) Principal component analysis demonstrating clear segregation between control and treated erythroid cell populations: the control cell population (red ellipse) and the 7,17-diHDPAn-3 cell population (blue ellipse). Each dot represents 1 biological RNA-seq replicate (n = 4 per group). (D) RT-qPCR analysis of hsp90aa1.1 and xpo1b expression using RNA extracted from whole embryos (biological triplicates with technical triplicates; 30 embryos per sample). (E) RT-qPCR analysis using RNA from FACS-isolated erythroid cells (40 000-50 000 cells pooled from 40 Tg(gata1:DsRed) embryos per sample). RT-qPCR was performed using three biological replicates, each with technical triplicates. Data are presented as mean ± SD. Statistical significance was determined using unpaired 2-tailed Student t tests. ∗P < .05; ∗∗P < .001. ctrl, control; DEG, differentially expressed genes; FC, fold change; ns, no significant difference; PC1, principal component 1.