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. 2015 Mar 5;4(4):658–669. doi: 10.1016/j.stemcr.2015.02.003

Figure 4.

Figure 4

Regulation of SMAD5 by L3MBTL1 Occurs in Erythroid Cells

(A) KD of L3MBTL1 induces early expression of the erythroid-specific marker GlyA in iPSC-derived CD34+ cells. Representative flow cytometry analysis of iPSCs after 2 days in Epo-induced culture.

(B) Expression levels of EKLF, NF-E2, LMO2, EPOR, and fetal globin gene were assessed in iPSC-derived erythroid progeny by qRT-PCR. Data were normalized by GAPDH expression. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.

(C) K562 erythroleukemia cells were retrovirally transduced to express L3MBTL1-HA (blue bars) or empty MIGR1 control (gray bars), crosslinked with 1% formaldehyde, and immunoprecipitated with an anti-SMAD5 antibody or IgG antibody. Primers covering the SMAD-binding motifs (Adelman et al., 2002) across the upstream enhancer, the proximal promoter, and the intronic enhancer of the EKLF were utilized. Data were normalized by 10% input. The data represent the mean ± SD of the three independent experiments.

(D) EKLF, NF-E2, GATA-1, LMO2, EPOR, and BCL11A expression levels were evaluated by qRT-PCR in L3MBTL1-KD CB CD34+ cells after 7 days of erythroid-supporting culture (100 ng/ml SCF and 6 U/ml EPO) compared to controls. GAPDH served as a housekeeping gene control. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.

(E) Fetal globin levels were evaluated in L3MBTL1-KD CB CD34+ cells by western blot. Tubulin served as the loading control.

(F) Overexpression of L3MBTL1 decreases globin gene mRNA levels in cord blood CD34+ cells, as shown by qRT-PCR. Cells were cultured for 3 days in liquid culture that supports erythroid differentiation. GAPDH served as the housekeeping gene. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.

(G) Overexpression of L3MBTL1 dramatically decreases protein expression levels of gamma globin in K562 cells, as shown by western blot assay. Tubulin served as the loading control. See also Figure S4.