Figure 2.
+6-kb enhancer regulates expression of Cebpe in murine myeloid cells. (A) Immunoblot with protein lysates from 32D cells stably transduced with empty vector (EV) or vector expressing dCas9-KRAB and sgRNAs targeting the +6-kb enhancer (sg1-6). Levels of α-tubulin (TUBA1A) were used as loading control. (B) Heat map shows genes that are significantly upregulated or downregulated in 32D cells stably expressing dCas9-KRAB-sg2 (sg2) or dCas9-KRAB-sg5 (sg5) compared with EV. (C) qRT-PCR validation of RNA-sequencing data for selected myeloid-specific genes downregulated in sg2- and sg5-transduced cells. (D) Venn diagram depicts the number of genes commonly downregulated between dCas9-KRAB sg2- and sg5-transduced 32D cells and immature neutrophils from Cebpe-KO mice. (E) qRT-PCR for Cebpe and its target genes (Ltf and Ngp) following G-CSF treatment of EV-, sg2-, and sg5-transduced 32D cells. Y-axis represents relative expression of genes normalized to Gapdh. **P < .01, *P < .05.

+6-kb enhancer regulates expression of Cebpe in murine myeloid cells. (A) Immunoblot with protein lysates from 32D cells stably transduced with empty vector (EV) or vector expressing dCas9-KRAB and sgRNAs targeting the +6-kb enhancer (sg1-6). Levels of α-tubulin (TUBA1A) were used as loading control. (B) Heat map shows genes that are significantly upregulated or downregulated in 32D cells stably expressing dCas9-KRAB-sg2 (sg2) or dCas9-KRAB-sg5 (sg5) compared with EV. (C) qRT-PCR validation of RNA-sequencing data for selected myeloid-specific genes downregulated in sg2- and sg5-transduced cells. (D) Venn diagram depicts the number of genes commonly downregulated between dCas9-KRAB sg2- and sg5-transduced 32D cells and immature neutrophils from Cebpe-KO mice. (E) qRT-PCR for Cebpe and its target genes (Ltf and Ngp) following G-CSF treatment of EV-, sg2-, and sg5-transduced 32D cells. Y-axis represents relative expression of genes normalized to Gapdh. **P < .01, *P < .05.

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