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 2. CypD inhibition attenuates programmed necrosis and apoptosis in MTB-infected human macrophages. The parental control human macrophages (“Pare”), with or without cyclosporin A (CsA) pretreatment (5 μM, for 1h), as well as the stale macrophages with the CypD shRNA (“shCypD”) or the lenti-CRISPR-Cas9 CypD knockout construct (“koCypD”), were infected with Mycobacterium tuberculosis (MTB) for applied time periods, CypD mRNA (A) and protein (B) expression was shown; Mitochondrial depolarization, cell viability, cell necrosis and apoptosis were tested by JC-1 staining (C), CCK-8 (D), medium LDH release (E), and Caspase-3/TUNEL assays (F and G) assays, respectively. The parental control human macrophages (“Pare”) as well as the stable macrophages with the CypD-expression construct (“OE-CypD”) or the empty vector (“Vec”) were infected with MTB for applied time periods, CypD mRNA (H) and protein (I) expression was shown; Mitochondrial depolarization (J), cell viability (K) and cell necrosis (L) were tested similarly. CypD expression was quantified, normalized to Tubulin (B and I). Data were presented as mean ± SD (n=5), and results were normalized to “C”. * P <0.05 vs. “C” treatment in “Pare” macrophages. #P <0.05 vs. MTB treatment in “Pare” macrophages. Experiments in this figure were repeated four times with similar results obtained.