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. 2016 Jan 11;11(1):e0146554. doi: 10.1371/journal.pone.0146554

Fig 6. NOS2-NO axis induces caspase-8 activation in M. fortuitum infected HKM.

Fig 6

(A) HKM were transfected or pre-treated separately with or without specific siRNA and indicated inhibitors respectively and at 24 h p.i. NOS2 protein (FITC conjugated) expression studied by confocal microscopy (×40). The images are representative of three independent experiments. (B) HKM were transfected or pre-treated separately with or without indicated siRNA or inhibitors respectively and at 24 h p.i. NO release measured. HKM treated with SNP was taken as positive control for the assay. (C) Fold change in NOS2 mRNA expression was determined in HKM transfected with specific siRNA or non targeted siRNA followed by M. fortuitum infection 12 h p.i. (D) Intracellular bacterial load was determined in HKM infected with M. fortuitum at indicated time p.i. or HKM transfected or pre-treated with indicated siRNA or inhibitors respectively 24 h p.i. by dilution plating on 7H11 agar plate. (E) Relative caspase-8 activity measured at 24 h p.i. in HKM pre-treated or transfected with indicated inhibitors or siRNA respectively. 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+MF+Sc; aP<0.05, compared to HKM+MF (5 h); bP<0.05, compared to HKM+MF (12 h). HKM, control head kidney macrophage; HKM+Sc, HKM transfected with scrambled siRNA; HKM+MF, HKM infected with M. fortuitum; HKM+MF+Sc, HKM transfected with scrambled siRNA infected with M. fortuitum; HKM+MF+siRNA-NOS2, HKM transfected with siRNA-NOS2 infected with M. fortuitum; HKM+MF+AMG, HKM+MF+LNAME, HKM+MF+U0126, HKM+MF+Z-IETD-FMK, HKM were pre-treated with AMG, LNAME, U0126 or Z-IETD-FMK, respectively followed by M. fortuitum infection.