The human SOD2 expression lentivirus was generated as described previously in our lab [15 (link)]. In order to generate human constitutive HIF1α (HIF1C) lentivirus, the human wild type HIF1α cDNA was obtained from Open Biosystems, and the HIF1α double mutants at P402(A) and P564(A) named as constitutive HIF1α (HIF1C) [25 (link)] were generated using the Site-directed Mutagenesis Kit from Promega. It was then amplified by PCR and subcloned into the pLVX-Puro vector (from Clontech) using restriction sites of BamH I and Xba1 with the following primers: Forward: 5’- tcga-ggatcc – atg gag ggc gcc ggc ggc gcg -3’ (BamH I) and Reverse: 5’- gcgc-tctaga- tca gtt aac ttg atc caa agc -3’ (Xba I), and the HIF1C or empty (CTL) lentivirus was expressed through Lenti-X™ Lentiviral Expression Systems (from Clontech) per manufacturers’ instructions. The virus for HIF1C and SOD2 expression, or related empty (EMP) virus was used to infect THP1 cells, the positive clones were selected by 10μg/ml puromycin, the single colony was picked up, and the expression efficiency for both SOD2 and HIF1C was confirmed by real time PCR and western blotting. The stable SOD2/HIF1C expression THP1 cells were used for in vivo mice xenograft tumor study.
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