c-MPL is required for the cytokine-independent growth of UT-7/TPO CALR Del52-expressing cells. (A) A schematic representation of the c-MPL knockdown experiment for UT-7/TPO cells expressing CALR Del52. RNA was collected from a separate experiment for fluorescence-activated cell sorting analysis and viability determination. (B) Validation of c-MPL knockdown by quantitative reverse-transcription-PCR. UT-7/TPO CALR Del52-expressing cells (uninfected) and other cells infected with lentivirus expressing the indicated shRNA were analyzed. The relative expression of c-MPL normalized to GAPDH expression in total RNA purified from infected and sorted cells was determined by quantitative reverse-transcription-PCR. The mean value ± SD from 3 replicates are depicted. (C) Determination of infection efficiency by fluorescence-activated cell sorting for the detection of the surrogate marker DsRed. The original UT-7/TPO cells, UT-7/TPO CALR Del52-expressing cells (uninfected), and other cells infected with lentivirus expressing the indicated shRNA were analyzed. CALR Del52-expressing cells were marked by GFP (Figure 1); lentivirus-infected cells were marked by DsRed. (D) The viability of shRNA-expressing cells 24 (open bar) and 72 (solid bar) hours after infection was determined by dye exclusion assay; the percentages of dead cells ± SD from 3 replicates are depicted. FCM, flow cytometry; O/N, overnight.