Fig 1: C. albicans induces pro-inflammatory cytokines in whole-brain and BV-2 cells. C57BL/c mice were challenged i.v. with 25,000 CFU of C. albicans after which whole brains were harvested at the indicated days for analysis by real-time quantitative PCR (RT-qPCR), western blot, or ELISA. a, b Nuclear factor kappa B (NF-?B) expression as assayed by RT-qPCR (a) or western blot (b; p65 subunit) over 14 days. c Densitometric analysis of the data from b. d–f IL-1ß, IL-6, and TNF cytokine levels from whole-brain homogenates as assessed by ELISA. g BV-2 cells were seeded for 6 h in 24-well plates (1 × 105 cells per well) and then incubated with C. albicans (200 viable cells per well) for 16 h after which secreted IL-1ß, IL-6, and TNF were quantitated by ELISA. h BV-2 cells were seeded same as above and incubated with lysates of C. albicans (equivalent 200 viable cells per well), irradiated C. albicans (200 cells per well), secreted aspartic proteases (SAP, 1 µM), or inhibited secreted aspartic proteases (1 µM) for 16 h and the same cytokines were quantitated by ELISA (n = 4. mean ± S.E.M, ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using two-tailed Student’s t-test (g) or one-way ANOVA (a, c–f, h) followed by Dunnett’s test for multiple comparison). Data are shown as representative of two independent experiments
Fig 2: Effect of Agm on proinflammatory cytokines gene expression and release by BV-2 cells. Schedule of Lps stimulation: 4 and 6 h for qPCR analysis; 24 h for TNF, IL-1 beta, and IL-6 protein levels measured in cell supernatants by ELISA. (a–c) Gene expression and protein levels of TNF (a), IL-1 beta (b), and IL-6 (c) Data are presented as mean ± SEM from three separate determinations. Results were analyzed by two-way ANOVA followed by Bonferroni’s post hoc tests, except IL-6 secretion, which was analyzed by Mann–Whitney test for comparing the mean values between two groups (Lps vs. LpsAgm) because Ctrl and Agm groups were not detectable (labeled n.d. on (c)). Significance levels shown inside the graphs: * p < 0.05 compared with the control group; # compared with Lps group.
Fig 3: Effect of BG on inflammatory macrophages stimulated with different concentrations of LPS (500 ng mL-1, 1 µg mL-1, 10 µg mL-1). A) Schematic diagram of various treatments of RAW and the collection of conditioned media. B) Expression of pro-inflammatory genes (TNF-alpha, IL-6, iNOS, IL-1 beta) in RAW after different treatments. (“LPS”: stands for incubation of RAW with LPS; “BG”: stands for incubation of RAW with LPS following pretreatment with BG extract liquid at 1:100 or 1:200 dilution ratio; “CK”: stands for RAW cultured with normal medium) C) Concentration of TNF-alpha in the culture media in RAW after different treatments. D) Expression of anti-inflammatory gene ARG in RAW after different treatments. (n = 3, * indicates p < 0.05, ** indicates p < 0.01)
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