Exogenous MN1 inhibits expression of CEBPA and its downstream targets in CD34⁺ cells and in ATRA-treated U937 cells. (A) GFP-expressing human CD34⁺-mock and MN1 cells were sorted 3 days after the last transduction and expanded for 3 more days in media supplemented with progenitor cytokines. Real-time RT-PCR analysis was used to determine the expression of the indicated genes. Results show the expression of each gene relative to endogenous HPRT (mean of triplicates ± SEM). **P = .009; ***P < .001. (B) U937-mock or MN1 cells were seeded (10⁵ cells/mL in 4 mL medium) 24 hours before addition of vehicle or 1 μM ATRA, and endogenous CEBPA expression of untreated (mock or MN1) as well as 8- and 24-hour ATRA-treated cells was determined using real-time RT-PCR (normalized to HPRT; mean of triplicates ± SEM). (C) Mock or MN1-U937 cells were treated with 1 μM ATRA for the indicated times, and expression of G-CSFR relative to endogenous HPRT was determined by real-time RT-PCR of RNA of untreated (mock) and treated cells (mock + ATRA; MN1 + ATRA; mean of triplicates ± SEM). (D) Expression of CD11b was detected using FACS of 16- or 24-hour 1 μM ATRA-treated U937-mock or MN1 cells. Controls are mock cells vehicle-treated for 24 hours. (E) Real-time RT-PCR analysis was performed to determine G-CSFR and miR-223 expression in 72-hour 1-μM ATRA-treated U937-mock and MN1 cells (endogenous HPRT or RNU6B expression was used to normalize G-CSFR or miR-223 expression, respectively; mean of triplicates ± SEM). *P = .012; **P = .009; ***P < .001. (F) Similarly, expression of the RAR/RXR downstream target RARβ was determined using real-time RT-PCR in the same RNA samples that were used in panel B (mean of triplicates ± SEM). (G) Real-time RT-PCR was performed to analyze RARβ and HPRT expression in HL60-mock and MN1 cells treated with 1 μM ATRA for the indicated times (mean of triplicates ± SEM).