Figure 4.

Imiquimod exerts its effect on Rps14-deficient anemic embryos via on-target activation of TLR7 (A-D) Ventral views of 6-dpf embryos stained with o-dianisidine, treated with PHS to induce hemolytic stress or with DMSO (A,C) or with PHZ and imiquimod (B,D). Imiquimod rescued stress-induced anemia. (F) Quantified as the normal/total ratio of embryos across 3 replicates at a concentration of 20 µM. (E) Effect of imiquimod analyzed across the dose range, analyzed by nonlinear regression. (G) Flow cytometric analysis of rps14^E8fs;Tg(itga2b:GFP) single embryos exposed to hemolytic stress and then treated with DMSO or imiquimod. Imiquimod enhanced the itga2b:GFP^lo cells, and the effect was most marked in the Rps14^+/− embryos, where there was a significant interaction between the drug and genotype. (H-M) Ventral views of 6-dpf embryos stained with o-dianisidine, treated to induce hemolytic stress (PHZ) and with DMSO (H,K), gardiquimod (I,L), or motolimod (J,M). Gardiquimod, but not motolimod, rescued the stress-induced anemia in Rps14^+/− embryos. (N) The gardiquimod rescue effect analyzed across dose range by nonlinear regression. Tlr7 knockout was validated using Miseq (O) and restriction enzyme digest with BplI (P), which digested only the WT. (Q) Knockout of Tlr7 abrogated the rescue of anemia by imiquimod. Original magnification x80.