Research Paper Volume 11, Issue 24 pp 12661—12673

Targeting cyclophilin-D by miR-1281 protects human macrophages from Mycobacterium tuberculosis-induced programmed necrosis and apoptosis

Figure 6. miR-1281 is ineffective in CypD-depleted human macrophages. The stale human macrophages with the lenti-CRISPR-Cas9 CypD knockout construct (“koCypD”) were infected with the lentivirus encoding pre-miR-1281 (“lv-pre-miR-1281”) or antigomiR-1281, with puromycin selection stable cells were established. The macrophages were then treated with Mycobacterium tuberculosis (MTB) infection for applied time periods, cell viability (CCK-8 assay, A), cell necrosis (medium LDH release assay, B), miR-1281 levels (C) and CypD protein expression (D) were tested. The stale macrophages with lv-pre-miR-1281 were further transfected with the construct encoding the 3’UTR-depleted CypD (“no UTR”), after 48h CypD mRNA and protein expression was tested (E); The macrophages were further infected with MTB for applied time periods, cell viability (F), cell necrosis (G) and miR-1281 expression (H) were examined. CypD expression was quantified, normalized to Tubulin (D and E). Data were presented as mean ± SD (n=5). * P <0.05 vs. “C” treatment. #P <0.05 (E and G). Experiments in this figure were repeated five times with similar results obtained.