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. 2009 Jan;71(1):240–252. doi: 10.1111/j.1365-2958.2008.06528.x

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Journal compilation © 2009 Blackwell Publishing

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Fig. 4 — A sod4Δ/Δ deletion in a sod5Δ/Δ background boosts ROS accumulation. A. Northern analysis of SOD4, SOD5 and ACT1 mRNA. The clinical C. albicans SC5314 strain was grown at 30°C, 37°C and 37°C plus 10% FCS. B. ROS measurement by luminol-dependent chemiluminescence at 37°C in 2.5 min intervals over a 90 min period [relative luciferase units (RLU) min⁻¹ per 1000 BMDMs]. Stimulation of BMDMs with either the wild-type (CA-IF100) strain or sod5Δ/Δ (CA-IF019), sod5Δ/Δ::SOD5 (CA-IF027), sod4Δ/Δsod5Δ/Δ (CA-IF039) and sod4Δ/Δsod5Δ/Δsod6Δ/Δ (CA-IF070) mutant strains (MOI 5:1) (a). Quantification of the total ROS release between 10 and 50 min (striped area) by calculating the area under the curve (MOI 5:1) and calculating the fold differences. The average of four independent experiments is presented. Infection with sod4Δ/Δsod5Δ/Δ yields 1.36 ± 0.18 times more ROS than by a sod5Δ/Δ strain. *P < 0.05, sod4Δ/Δsod5Δ/Δsod6Δ/Δ triple mutant yields 1.43 ± 0.09 times more ROS than by a sod5Δ/Δ strain. *P < 0.02; and the sod4Δ/Δsod5Δ/Δsod6Δ/Δ triple mutant yields 1.09 ± 0.1 times more ROS than sod4Δ/Δsod5Δ/Δ. ***P > 0.09 (b). C. Superoxides measurement by lucigenin-dependent chemiluminescence at 37°C over a 90 min period [relative luciferase units (RLU) under the curve]. Stimulation of BMDMs with either zymosan (20 μg/well), the wild-type (CA-IF100) strain, the sod5Δ/Δ (CA-IF019), sod4Δ/Δsod5Δ/Δ (CA-IF039) and sod4Δ/Δsod5Δ/Δsod6Δ/Δ (CA-IF070) mutant strains (MOI 5:1). A and B. Results of one experiment per condition are shown. All data were reproduced in two independent experiments. D. Intracellular ROS production in response to wild-type (CA-IF100) strain or sod4Δ/Δ (CA-IF015), sod4Δ/Δsod5Δ/Δ (CA-IF039) mutant strains (MOI 5:1) or zymosan (100 μg ml⁻¹). ROS was measured by FACS analysis using H₂DCF-DA-staining of BMDMs after 45 min of infection. A–C. Results of one experiment per condition are shown. All data were reproduced in at least three independent experiments. Statistical significances were calculated using a two-tailed Student's t-test.