Figure 7.

Figure 7.

The negative regulatory role of TMEM187 in erythroid differentiation was verified in zebrafish and mouse models. (A-C) Representative images of o-dianisidine staining of hemoglobin at 48 hpf in WT or tmem187^–/– zebrafish in ventral view (A) and profile view (B), quantified in (C). n = 19/group. (D) Validation of Myc-tagged human TMEM187 expression, detected by immunoblotting, in BM cells of TMEM187^Tg(loxp/–) (Control) and Vav-iCre TMEM187^Tg(loxp/–) (cKI) mice. (E-F) Size (E) and body weight (F) comparison of the control and cKI mice. (G) Routine blood examinations: number of RBCs, hemoglobin concentration, and mean corpuscular volume. n = 4 for control group; n = 3 for cKI group. (H) Wright-Giemsa staining of peripheral smear. (I) Percentage of Ter119⁺ erythroblasts from the BM of the control and cKI mice, quantified in (J). (K) Frequency of erythroblast populations among Ter119⁺ cells in the BM of the 2 genotype mice, quantified in (L). n = 3 for control group; n = 4 for cKI group. (M) Schematic of the experimental timeline (top). Control and cKI mice were subjected to repeated blood withdrawal (300 μL per day) for 3 days. Peripheral blood samples were collected at the indicated time points (days 0, 4, 7, 10, 14) for analysis. (N) Dynamics of RBC, hemoglobin concentration, and hematocrit in control (blue line) and cKI (red line) mice. (O) Prussian blue staining of BM smears. Arrows point to iron-stained positive areas. (P) Nonheme total iron content in BM, revealed by ferrozine assays. n = 4 for control group; n = 4 for cKI group. (Q-R) Expression of erythroid differentiation-related proteins (Q) and iron metabolism-related proteins (R) in Ter119⁺ cells derived from BM of the 2 genotype mice, detected by immunoblotting. Ponceau S stains were used as internal controls. n = 3 for control group; n = 3 for cKI group. (S) The proposed mechanism by which TMEM187 regulates erythroid differentiation, maturation, and senescence through interaction with RAB11A to compete with RAB11A-and-GRAB interaction to affect TfR1 trafficking. t test was used for significance. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. FSC, forward scatter; hpf, hours post fertilization; ns, no significance; #, nonspecific band.