shRNA-mediated silencing of RINF imparts resistance to ATRA-induced terminal differentiation of NB4 cells. (A) Lentiviral shRNA vectors and their mRNA target sequences used to knock down RINF expression. After infection and selection of NB4 cells (see “Methods”) with the lentiviral vector constructs, their efficiencies to target basal RINF expression were monitored by quantitative RT-PCR measuring basal RINF mRNA expression levels (indicated in percent of mock control, the pLKO.1/empty vector). The most efficient knockdowns were obtained with shRNA/RINF-3 and shRNA/RINF-4 constructs (61% and 85%, respectively, in the absence of ATRA) indicated in red. (B) Cell growth (population doublings) of NB4 cells, stably expressing the shRNA constructs, in the presence of 1 μM ATRA. The presented kinetic experiment (here during 12 days of ATRA treatment) is representative of 3 separate experiments (performed from different batches of infections). † indicates postmaturation cell death of control cells. (C) RINF mRNA expression was assessed by quantitative RT-PCR (percent of untreated mock control at 12 hours of culture), terminal differentiation was assessed by cell morphology at day 4 (scale bar, 25 μm), and NBT reduction assay was assessed at day 2 (scale bar, 25 μm) of NB4 cells infected with empty, shRNA/scramble, shRNA/RINF-3, and shRNA/RINF-4 vectors. Cells were treated or not treated with 1 μM ATRA for 4 days (first round of ATRA, d0-4, left panel). After 2 more weeks of culture in the absence of ATRA, shRNA/RINF-3 and shRNA/RINF-4 cells that escaped the first round of ATRA (indicated by an arrow [→]) were retreated (second round of ATRA, days 20 to 24, right panel) for 4 days with 1 μM ATRA.