Journal: PLOS Neglected Tropical Diseases

Article Title: Giardia duodenalis MIF induces host intestinal damage via CD74 receptor mediated NLRP3 inflammasome activation

doi: 10.1371/journal.pntd.0013968

Figure Lengend Snippet: (A) PMϕs were incubated with 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL rGdMIF or G. duodenalis (MOI = 3) for 24 h. The expression of total and phosphorylated proteins of IκB and P65 were detected by western blot. (B. C) The densitometric analysis of P-IκB and P-P65 protein. All results were representative of three number of biologically independent experiments (n = 3). Data was shown as the mean ± SD, statistical significance was assessed using one-way ANOVA. n.s. p > 0.05 indicates not significant, * p < 0.05, ** p < 0.01. (D) Spearman correlation between P-IκB, CD74, NLRP3, IL-1β and GSDMD-NT. (weak correlation: 0.1 < r < 0.3, moderate correlation: 0.3 < r < 0.5, strong correlation: 0.5 < r < 1.0). (E-H) PMϕs were transfected with control-siRNA (sicontrol) and CD74-siRNA (siCD74) for 24 h, and then stimulated with rGdMIF (1 μg/mL) for 24 h. The expression of CD74, total and phosphorylated proteins of IκB and P65 were detected by western blot and densitometric analysis. All results were representative of three number of biologically independent experiments (n = 3). Data was shown as the mean ± SD, statistical significance was assessed using one-way ANOVA and unpaired Student’s t-test. n.s. p > 0.05 indicates not significant, * p < 0.05, *** p < 0.001 and **** p < 0.0001. (I-M) PMϕs were pretreated with BAY11-7082 (5 μM) for 2 h and then stimulated with rGdMIF (1 μg/mL) for 24 h. The expression levels of P-IκB, NLRP3-GSDMD pathway related proteins were detected by western blot and densitometric analysis. (N) NLRP3 (Red) was observed by IFA, scale bar: 10 μm. Three fields of view were analyzed in total per sample, and three images of view were captured in total per sample, quantitative analysis of the presence of NLPR3 signals in x% of the cells in each image was performed using Image J software. (O) Cell death was detected by LDH assay. All results were representative of three number of biologically independent experiments (n = 3). Data was shown as the mean ± SD, statistical significance was assessed using unpaired Student’s t-test. n.s. p > 0.05 indicates not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: PMφs were incubated with 1 μg/mL His, 1 μg/mL rGdMIF, or G. duodenalis (MOI = 3) for 24 h; PMφs were transfected with control-siRNA (sicontrol) and CD74-siRNA (siCD74) using Lipo2000 and cultured for 24 h, and then stimulated with rGdMIF (1 μg/mL) for 24 h; PMφs were pretreated with BAY11–7082 (5 μM, Cat. No. HY-13453, MCE) for 2 h and then stimulated with rGdMIF (1 μg/mL) for 24 h. The cells were then fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100, both at RT for 20 min.

Techniques: Incubation, Expressing, Western Blot, Transfection, Control, Software, Lactate Dehydrogenase Assay