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. 2015 Jul 31;290(37):22805–22817. doi: 10.1074/jbc.M115.659151

FIGURE 5.

FIGURE 5.

Effects of SOD2 knockdown on ROS production in activated microglia. After transfection of SOD2 siRNA (siSOD2) or control siRNA (siCon) into primary microglia, the cells were incubated for 24 h and then further incubated in the presence or absence of 10 ng/ml LPS for up to 24 h. A, after 6 h of incubation, the mRNA levels of SOD2 were measured using real-time PCR and are represented as -fold changes from the levels measured in untreated cells. The values represent the mean ± S.E. (error bars) of six separate experiments. The data were compared using Student's t test with Holm's corrections for multiple comparisons. **, p < 0.01 versus the untreated group carrying control siRNA. ##, p < 0.01 versus the LPS-treated group carrying control siRNA. B, after incubation for 24 h, SOD2 proteins in the cell lysates were detected using immunoblotting. The results are representative of four independent experiments. C, the band intensity represented in B was determined using the ImageJ software program. The values represent the mean ± S.E. of four separate experiments. The data were compared using Student's t test with Holm's corrections for multiple comparisons. **, p < 0.01 versus the untreated group carrying control siRNA; ##, p < 0.01 versus the LPS-treated group carrying control siRNA. D, representative images of ROS inside cells measured using DHE 3 h after stimulation with 10 ng/ml LPS. A cell membrane-permeable SOD, SOD-PEG, was added just before the addition of LPS, at a final concentration of 100 units/ml. The results are representative of five independent experiments. Bar, 100 μm. E, SOD2 siRNAs (siSOD2) or their control siRNAs (siCon) were transfected into primary microglia. After 24 h of incubation, the cells were pretreated with the NADPH oxidase inhibitors, 1 μm DPI or 100 μm apocynin (Apo), and then cultured in the presence or absence of 10 ng/ml LPS for 3 h. The amount of superoxide in the culture media was measured using acetylated cytochrome c. The values represent the mean ± S.E. of four separate experiments. The data were analyzed using one-way ANOVA, followed by Student's t test or Dunnett's test. The Bonferroni method was used to correct for multiple comparisons. *, p < 0.05 versus the untreated group carrying control siRNA; ##, p < 0.01 versus the LPS-treated group carrying control siRNA. a, p < 0.05; aa, p < 0.01 versus the LPS-treated group carrying SOD2 siRNA.