Transmembrane Protein ANTXR1 Regulates γ-Globin Expression by Targeting the Wnt/β-Catenin Signaling Pathway

Figure 2

Effects of ANTXR1 overexpression or knockdown on γ-globin mRNA and protein levels. (a, b) ANTXR1 was overexpressed using the PHAGE-fEF-1a-IRES-ZsGreen-2 vector in K562 cells, while cells transfected with the empty vector served as controls. ANTXR1 and γ-globin mRNA levels were measured by qRT-PCR. (c) In K562 cells, ANTXR1-sh1-5 was separately inserted into the pSIH1-H1-copGFPshRNA vector to test the knockdown efficiency of ANTXR1. Of the five shRNA vectors, ANTXR1-sh1 and ANTXR1-sh5 had the most significant knockdown efficiencies of 82% and 88%, respectively. (d) After the K562 cells were infected with ANTXR1-sh1, ANTXR1-sh5, and control shNC, respectively, the mRNA levels of ANTXR1 and γ-globin were determined using qRT-PCR. ANTXR1-sh5 had a greater effect on γ-globin expression than ANTXR1-sh1. (e) Western blot analysis was used to detect ANTXR1 and γ-globin levels after infection of K562 cells with ANTXR1 overexpression vectors and interference vectors (ANTXR1-sh1 and ANTXR1-sh5). (f) At days 0, 4, and 7 of erythroid differentiation after infection of HUDEP-2 cells with ANTXR1-sh5 and shNC vectors, ANTXR1,γ-globin, and β-globin expression was determined by western blotting. (g, h) After infection of umbilical cord blood CD34⁺ cells with overexpression of the ANTXR1 vector, the mRNA levels of ANTXR1 and γ/γ+β globin ratio at days 11, 14, and 16 of erythroid differentiation were determined by qRT-PCR. (i, j) After infection of umbilical cord blood CD34⁺ cells with ANTXR1-5 and shNC vectors, the mRNA levels of ANTXR1 and γ/γ+β globin ratio at days 11, 14, and 16 of erythroid differentiation were determined by qRT-PCR. (k) After infection of umbilical cord blood CD34⁺ cells with the ANTXR1 overexpression vector and interference vector, the protein levels of ANTXR1,γ-globin, and β-globin at days 11, 14, and 16 of erythroid differentiation were detected by western blotting. (l) Umbilical cord blood CD34⁺ cells interfered with ANTXR1 and were cultured under conditions that promoted erythroid maturation. The cells were collected at days 11, 14, and 16, respectively. Flow cytometry was used to detect HbF-immunostained cells (F cells). These experiments were repeated three times. The error bar represents the SD. and . NC designates the negative control.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)