Lentiviral vectors

GIPZ HIF-1α or control shRNA plasmids were from Open Biosystems. Lentiviral vectors were obtained by HEK-293T transfection as previously described [55]. MOLM-13, THP-1 and Kasumi-1 cells were transduced by spinoculation, selected with puromycin (4 μg/mL, 7 μg/mL and 1 μg/mL respectively) and sorted for GFP expression 2 weeks after transduction (MoFlo XDP, Beckman Coulter), leading to a bulk of cells with different integrations and GFP levels.

For in vivo experiments, shCTRL and shHIF-1α MOLM-13 cells were further transduced with a bidirectional lentiviral vector co-expressing luciferase and truncated nerve growth factor receptor (ΔNGFR) (kindly provided by G. Escobar, B. Gentner and L. Naldini, unpublished data) and sorted for ΔNGFR (CD271, PE, BD Pharmingen) expression 2 weeks after transduction.

For in vivo experiments with LNA-ON (EZN-3088 and EZN-2968), MOLM-13 cells transduced with the bidirectional lentiviral vector co-expressing luciferase and ΔNGFR and sorted for ΔNGFR expression 2 weeks after transduction, were electroporated with EZN-3088 and EZN-2968 and injected in mice after 24 hours.