STMN4 Antibody - #DF4547

Figure 4 Cu overload destroys embryonic HSPC cytoskeleton (A) Schema for the experiments in (B) and (C). (B) Enriched GO terms for down-regulated DEGs in Cu-stressed HSPCs at 33 hpf. (C) Cytoskeleton protein immune-fluorescence for Cu-stressed HSPCs at 33 hpf. C1, C4, anti-β-tubulin staining; C2, C5, DAPI staining; C3, C6, merged; C7, the percentage of the Cu-stressed runx1GFP+ cells exhibiting destructive cytoskeleton; C8, calculation of the fluorescence intensity in each cell. (D) Mitotic malformation of HSPCs in the cytoskeleton gene morphants (tuba1a-MO, stmn4-MO, tubb5-MO, and tmsb2-MO together) at the 33 hpf (AGM) and 72 hpf (CHT), respectively. D1, D5, D10, D14, DAPI staining; D2, D6, D11, D15, anti-α-tubulin staining; D3, D7, D12, D16, anti-GFP staining; D4, D8, D13, D17, merged. At least 10 mitotic HSPCs in more than 10 embryos were observed for each group. D9, D18, calculation of the length of spindles in metaphase runx1GFP+ cells at 33 hpf and 72 hpf, respectively. (E) The protein level of Tuba1a (E1) and Stmn4 (E3) in tuba1a mutants and stmn4 mutants at 33 hpf, respectively. GAPDH was used as an internal control. (F) Phenotypes of tuba1a (F2, F5) mutants with a 4-bp deletion and stmn4 (F3, F6) mutants with an 8-bp deletion at 33 hpf and 120 hpf, respectively. (G) Expression of runx1 and cmyb in tuba1a mutants with or without Cu stresses at 72 hpf. G9, calculation of runx1 and cmyb expression in embryos from different groups. (H) Expression of runx1 and cmyb in stmn4 mutants with or without Cu stresses at 72 hpf. H9, calculation of runx1 and cmyb expression in embryos from different groups. F1-F6, G1-G8, H1-H8, lateral view, anterior to the left, and dorsal to the up. Data are mean ± SD. t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. NS, not significant. Scale bars, 1.2 μm (C1-C6), 2 μm (D1-D8, D10-D17), 100 μm (D1-D3, F1-F6), and 200 μm (G1-G8, H1-H8).

Fig. 1 Stmn4 deficiency led to eye morphological defects. (A) Copper (Cu) stress induced obviously reduced expression of stmn4 in eyes during zebrafish embryogenesis (A1 − A4), cross-section of the eye (A5, A6) and the calculation of the relative expression levels of stmn4 (A7). (B) Western blot detection of Stmn4 protein levels in WT and stmn4−/− at 24 hpf and 48 hpf (B1 − B2), and the mRNA levels of stmn4 in WT and stmn4−/− at 24 and 48 hpf (B3), respectively. (C) Embryonic and eye development in stmn4−/− and WT embryos and larvae at different developmental stages (C1 − C12), and the quantification of the eye size in WT and stmn4−/− embryos and larvae (C13), respectively. (D) HE staining showed the difference in the eyes of WT and stmn4−/−embryos at 48 hpf (D1 − D4), and the calculation data (D5, D6). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. A1, A2, lateral view, anterior to the left, and dorsal to the up; A3, A4, head to the up, and dorsal to the down; C1 − C12, lateral view, anterior to the up, and dorsal to the right. Scale bar, 200 μm (A1 − A4), 100 μm (A5, A6, C1 − C12, D1, D2), 50 μm (D3, D4). ***P