Puro ires gfp

Leukemia cell lines were obtained from ATCC and DSMZ. Leukemia cell lines were grown in RPMI supplemented with FCS 10% and penicillin-streptomycin. The media for SKNO-1 also included GM-CSF 10 ng/mL. The media for 32D cells included murine IL-3 1 ng/mL. HEK293T and 3T3 cells were grown in DMEM supplemented with FCS 10% and penicillin-streptomycin. GFP expressing K562 leukemia cells were generated by transducing K562 cells with MSCV PIG (Puro-IRES-GFP; Addgene plasmid #18751) retrovirus and selecting with puromycin.

For RNAi knockdown studies, pMSCs were cultured to 50–60% confluency in 100-mm petri dishes and transfected with 30 nM of JMJD6 Silencer^® siRNA duplexes (ID:23290, Cat # 4392420, Thermo Fisher Scientific, Waltham, MA, United States) or Silencer^® Negative Control siRNA (Cat #4390844, Thermo Fisher Scientific) using jetPRIME^® buffer (Polyplus transfection,^TM Illkirch, France) for 24 h. For overexpression studies, the p6352 MSCV-CMV-CMV-Flag-HA-JMJD6 plasmid [Addgene, Cambridge, MA, United States; Plasmid 31358 (Rahman et al., 2011 (link))] was obtained as previously described (Alahari et al., 2015 (link)). Mutant JMJD6 plasmids were generated by site-directed mutagenesis against JMJD6 WT plasmids by mutating histidine 187 and aspartic acid 189 to alanine (H187A and D189A, respectively) (Alahari et al., 2018 (link)). Empty Vector control (EV) consisted of a MSCV PIG (Puro IRES GFP) (Plasmid 18751, Addgene) plasmid on an empty vector backbone. Plasmid (0.5 μg or 1.0 μg) was used for transfection studies in pMSCs. All plasmids were validated by sequencing.