Suz12, Dicer1, and Ago1 knockdowns. HL60 cells, wild-type (wt), were infected with empty lentiviral vectors (pgk or pGIPZ) or with lentiviral constructs expressing siRNAs against Suz12 (siSuz12#1, siSuz12#6), Dicer1 (siDicer1), and Ago1 (siAgo1). (A) Suz12 product as measured by immunoblot analysis with β-tubulin as loading control. (B) Quantitative RT-PCR of NFI-A mRNA levels in indicated cells treated or not (CTR) with 1μM RA for 48 and 72 hours. Bottom panel: RT-PCR measuring the NFI-A mRNA levels and myeloid differentiation marker TGase-II. GAPDH levels were used as RNA loading control. (C) ChIP assays performed using an anti-H3K27me3 antibody and RT-PCR primers encompassing NFI-A gene region 1 at the indicated times in cells treated or not (CTR) with RA 1μM. Rabbit control IgG was used as nonspecific antibody. Bottom panel: Enrichment of the H3K27me3 on NFI-A promoter region 1 as measured by quantitative RT-PCR. Error bars represent SD of 3 independent evaluations. Statistical significance was calculated with respect to RA-treated pgk cells. *P < .05. **P < .01. (D) Percentage of CD11b-positive cells in cells treated (RA) or not (CTR) at the indicated times and concentrations of RA, as measured by flow cytometry. Statistical significance was calculated with respect to RA-treated pgk cells. *P < .05. **P < .01. (E) Immunoblotting analysis of TGase-II, an RA target gene involved in granulocytic differentiation.34 β-tubulin is a loading control. (F) Immunoblotting analysis of Dicer1 and Ago1 products. β-tubulin as loading control. (G) Quantitative RT-PCRs measuring miR-223 and NFI-A mRNA levels and the percentage of CD11b-positive cells as measured by flow cytometry in pGIPZ (●), siDicer1 (■), and siAgo1 (▾) cell lines, after 72 hours of treatment with the indicated concentrations of RA. Statistical significance was calculated with respect to RA-treated pGIPZ cells. *P < .05. **P < .01. (H) ChIP assay and H3K4me3 and H3K27me3 chromatin mark enrichments on NFI-A promoter region 1 as measured by quantitative RT-PCR. Statistical significance was calculated with respect to RA-treated pGIPZ cells. *P < .05. **P < .01. Results represent the average of 3 independent evaluations ± SD. Bars represent the average of 3 independent evaluations ± SD.
