Figure 5.

RNA sequencing analysis of HSPCs shows the effects on WNT and inflammatory signaling pathways. (A) Representative western blot of p-Eif2α (phosphoserine 51) and Eif2α total in Rp14 mutants at 6-dpf exposed to PHZ and/or imiquimod. (B) Normalized ratio (to untreated WT) of p-Eif2α/total Eif2α shown in 3 experiments, represented by different colored lines. The P-value refers to the effect of genotype on the ratio. (C) Differential gene expression of Rps14^+/− vs Rps14^+/+ embryos shown as a volcano plot for all conditions combined. Rps14 is shown in bold. (D) Metascape Pathway Analysis of Rps14^+/− vs Rps14^+/+ differentially expressed genes showing the top 20 enriched pathways. (E-F) GSEA analysis comparing Rps14^+/− with Rps14^+/+ in DMSO-treated vs imiquimod-treated HSPCs. (E) Negative regulation of WNT signaling was enriched in Rps14^+/− vs Rps14^+/+ DMSO-treated HSPCs, and the effect was reversed in imiquimod-treated HSPCs. (F) Similarly, inflammatory signaling was enriched in Rps14^+/− vs Rps14^+/+ DMSO-treated HSPCs but suppressed in imiquimod-treated HSPCs. (G) Quantification of total fluorescence of NFKβ/GFP at 4 dpf shows an increase with PHZ stress but a decrease with imiquimod in Rps14^+/− compared with Rps14^+/+. (H) PHZ causes an increase in il1b expression by in situ hybridization in Rps14^+/− which is rescued by imiquimod. Statistical comparisons are by 2-way ANOVA (B,H; genotype and condition) or ANOVA (G).