Figure 5. Mitapivat-treated Hbb^th3/+ mice showed reduced ferroportin, reduced duodenum iron overload, and downregulation of the HIF2α–Dmt1 signaling axis.

(A) Iron staining (Perl’s Prussian blue) in duodenums from WT and Hbb^th3/+ mice treated with vehicle or mitapivat. Original magnification ×400. Scale bars: 100 μM. One representative image from 4 with similar results. Arrows indicate iron deposits. (A, right panel) Quantification of iron staining. Data are mean ± SD (n = 5). *P < 0.05 compared with WT or ^#P < 0.05 compared with vehicle-treated Hbb^th3/+ mice by 2-tailed t test. (B, upper panel) Western blot analysis using specific antibodies against PKR and PKM2 of duodenums from WT and Hbb^th3/+ mice treated with vehicle or mitapivat, GAPDH as loading control. One representative gel from 4 with similar results. (B, lower panel). Densitometric analyses of the Western blots, mean ± SD (n = 6). *P < 0.05 compared with WT by 1-way ANOVA with Bonferroni multiple comparison correction. ^#P < 0.05 compared with vehicle-treated Hbb^th3/+ mice by 1-way ANOVA with Bonferroni multiple comparison correction. (C, left panel) Western blot analysis with specific antibodies against HIF2α, pNF-κB p65, and NF-κB p65 of duodenums from WT and Hbb^th3/+ mice treated with vehicle or mitapivat, GAPDH as loading control. One representative gel from 4 with similar results. (C, right panel) Densitometric analyses of the Western blots, mean ± SD (n = 6). *P < 0.05 compared with WT or ^#P < 0.05 compared with vehicle-treated Hbb^th3/+ mice by 1-way ANOVA with Bonferroni multiple comparison correction. (D) mRNA expression of Dmt1-IRE by qRT-PCR on duodenums from WT and Hbb^th3/+ mice treated with vehicle or mitapivat. Experiments were performed in triplicate. Data are mean ± SD. ^#P < 0.02 compared with WT mice and *P < 0.05 compared with vehicle-treated mice by 2-way ANOVA with Tukey’s multiple comparisons test.