Supplementary Figure 4 from Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on <i>MIR300</i> Antiproliferative and PP2A-Activating Functions

Figure S4. MIR300 anti-proliferative activity accounts for BMM-induced LSC entry into quiescence. A, Structure of 14q32 DLK1-DIO3 genomic imprinted locus hosting the MEG3-regulated human MIR300. B, MIR300 levels in 5-Aza- or DMSO-treated (24h) Ph+ cells. C, Effect of hypoxia on proliferation of CFSE+CD34+ CML-BC cells. D, left: Effect of MSC (HS-5)-derived CM on LAMA-84 proliferation expressed as fold changes of CFSE mean of fluorescence intensity (MFI)+/-SEM; middle: pro-apoptotic effect of PAD (FTY720; 2.5uM) and DMSO (control) on HS-5-cultured LAMA-84 cells; right: Effect of MSC (HS-5)-derived CM BCR-ABL1 expression (anti-ABL1) and activity anti-PY), phospho-BCR-ABL1, JAK2 expression and activity JAK2 Y1007/1008, PP2A activity (pPP2AY307 inactive form) and GRB2 used as a control (blots are representative of three independent experiments). E, Levels of C/EBPbeta and GRB2 mRNA and protein in HS-5 cells exposed to hypoxia (48h; 1% O2). F, Effect of neutralizing TGFbeta antibody (anti-TGFb Ab; 48h, 1.25 μg/ml) on MIR300 levels in CD34+ CML-BC cells. G, Effect of ectopic C/EBPalpha (MigR1-deltauORF-C/EBPalpha-HA) and C/EBPbeta (MigR1-C/EBPB-ERTAM) on MIR300 levels in K562 cells. Immunoblot shows levels of C/EBPbeta and GRB2 in normoxic and hypoxic K562 cells.