Figure 6.
The regulatory networks of lncRNAs, TFs, and genes during terminal erythroid differentiation. (A) The lncRNAs-TF-gene regulatory network in EB: specific lncRNA identified in EB (left column); TFs expressed in EB (middle column); and hub genes in EB (right column). The line between the columns represents the possible regulatory relationship among factors. (B) Detection of CD235a+ cells in differentiated CD34+ cells on day 11 by flow cytometry in green fluorescent protein-positive cells with PCED1B-AS1-KD. The percentage on right in each figure represents the proportion of CD235a+ cells. (C) Relative expression of erythroid-specific genes in differentiated CD34+ cells with PCED1B-AS1-KD on day 12 detected by qPCR assay. (D) Colony-forming capacity analysis of PCED1B-AS1 knockdown in CD34+ cells. (E) Morphology of differentiated CD34+ cells on day 11 (original magnification ×100; Wright-Giemsa stain): vector control (left) and PCED1B-AS1-KD (right). (F) GATA1 binding sites around PCED1B-AS1 locus in EB stage from ENCODE database (ENCFF957CWW). The binding sites are named Rank 1 to 4 from left to right. Rank3 locates in PCED1B-AS1 locus, and the other 3 binding sites locate outside the PCED1B-AS1 locus. (G) GATA1 ChIP-qPCR analyses of IgG and IP on Day 7 and Day 10. The GATA1 binding signal was detected at the 4 regions (Rank1, Rank2, Rank3, and Rank4). (H) Detection of activity of PCED1B-AS1 promoter without any changes or with deletion, mutation on GATA1 binding motif by dual luciferase reporter assay. PGL4.10-PCED1B-AS1-P represents the promoter of PCED1B-AS1 included in the construct of PGL4.10; PGL4.10-PCED1B-AS1-P-del represents the deletion of GATA1 binding motif in the promoter; PGL4.10-PCED1B-AS1-P-mut represents the mutation of GATA1 binding motif in the promoter. (I) Silver staining of the co-precipitated proteins with lncRNA PCED1B-AS1 in the in vitro RNA pull-down assay. Cytoskeleton protein or associated proteins indicated by asterisk (*). Statistical results were analyzed by Student t-test and Kruskal-Wallis test; *P < .05; **P < .01; ***P < .001. Ctrl, control group; KD, knockdown; OE, overexpression.

The regulatory networks of lncRNAs, TFs, and genes during terminal erythroid differentiation. (A) The lncRNAs-TF-gene regulatory network in EB: specific lncRNA identified in EB (left column); TFs expressed in EB (middle column); and hub genes in EB (right column). The line between the columns represents the possible regulatory relationship among factors. (B) Detection of CD235a+ cells in differentiated CD34+ cells on day 11 by flow cytometry in green fluorescent protein-positive cells with PCED1B-AS1-KD. The percentage on right in each figure represents the proportion of CD235a+ cells. (C) Relative expression of erythroid-specific genes in differentiated CD34+ cells with PCED1B-AS1-KD on day 12 detected by qPCR assay. (D) Colony-forming capacity analysis of PCED1B-AS1 knockdown in CD34+ cells. (E) Morphology of differentiated CD34+ cells on day 11 (original magnification ×100; Wright-Giemsa stain): vector control (left) and PCED1B-AS1-KD (right). (F) GATA1 binding sites around PCED1B-AS1 locus in EB stage from ENCODE database (ENCFF957CWW). The binding sites are named Rank 1 to 4 from left to right. Rank3 locates in PCED1B-AS1 locus, and the other 3 binding sites locate outside the PCED1B-AS1 locus. (G) GATA1 ChIP-qPCR analyses of IgG and IP on Day 7 and Day 10. The GATA1 binding signal was detected at the 4 regions (Rank1, Rank2, Rank3, and Rank4). (H) Detection of activity of PCED1B-AS1 promoter without any changes or with deletion, mutation on GATA1 binding motif by dual luciferase reporter assay. PGL4.10-PCED1B-AS1-P represents the promoter of PCED1B-AS1 included in the construct of PGL4.10; PGL4.10-PCED1B-AS1-P-del represents the deletion of GATA1 binding motif in the promoter; PGL4.10-PCED1B-AS1-P-mut represents the mutation of GATA1 binding motif in the promoter. (I) Silver staining of the co-precipitated proteins with lncRNA PCED1B-AS1 in the in vitro RNA pull-down assay. Cytoskeleton protein or associated proteins indicated by asterisk (*). Statistical results were analyzed by Student t-test and Kruskal-Wallis test; *P < .05; **P < .01; ***P < .001. Ctrl, control group; KD, knockdown; OE, overexpression.

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