Fig. 4. Activation of calpain/caspase-12 axis is consequent to ER-stress in M. fortuitum infected HKM.

a HKM were infected with M. fortuitum and at indicated time p.i. CAPNS1-mRNA expression was quantified by qPCR. b HKM were transfected separately with CAPNS1-siRNA or scrambled siRNA and CAPNS1-mRNA expression was quantified by qPCR. c HKM were infected with M. fortuitum and at indicated time p.i. calpain activity was measured using assay kits. d HKM transfected separately with CAPNS1-siRNA, scrambled siRNA or pre-treated with indicated inhibitors were infected with M. fortuitum and calapin activity measured at 2 h p.i. e HKM pre-treated with indicated inhibitors or transfected with CAPNS1-siRNA were infected with M. fortuitum and caspase-12 activity was studied at 24 h p.i. using specific kit and observed under confocal microscope ( × 40). The images are representative of three independent experiments. Vertical bars represent mean ± SE (n = 3).*P < 0.05, compared to HKM; γP < 0.05, compared to HKM + Sc; ^#P < 0.05, compared to HKM + MF; •P < 0.05, compared to HKM + Sc + MF. HKM, control head kidney macrophage; HKM + Sc, HKM transfected with scrambled siRNA; HKM + MF, HKM infected with M. fortuitum; HKM + Sc + MF, HKM transfected with scrambled siRNA followed by M. fortuitum infection; HKM + CAPNS1-siRNA + MF, HKM transfected with CAPNS1-siRNA then infected with M. fortuitum; HKM + PD150606 + MF, HKM + Calpain1i + MF, HKM + Calpain2i + MF, HKM + 4-PBA + MF, HKM + BAPTA/AM + MF, HKM + Z-ATAD-FMK + MF, HKM pre-treated with PD150606, Calpain1i, Calpain2i, 4-PBA, BAPTA/AM, Z-ATAD-FMK respectively and infected with M. fortuitum