Figure 4.

IFNγ Receptor Signaling Enhances but is not Required for CASP11 Inflammasome Activation

(A, C, and D) WT and Ifngr1−/− BMDMs were primed for 16 h overnight with the following treatments: unprimed (N/A), IFNγ (100 U/mL), IFNβ (100 U/mL), or LPS (10 ng/mL). CASP11 inflammasome activation was triggered by LPS (E. coli 0111:B4, 25 μg/mL) transfection with FuGENE HD.

(A) At 2 h following inflammasome activation, supernatants were collected to measure release of LDH for percent cell death calculations.

(B) Cell lysates were collected from BMDMs treated for 16 h of priming as described above and separated by SDS-PAGE. Western blot analysis was performed to determine protein expression of CASP11, GSDMD, and the loading control ACTIN. Molecular weight marker positions are shown to the left of each blot.

(C and D) Cell death kinetics were monitored over time following inflammasome activation by measuring the incorporation of SYTOX Green. (C) Comparisons between different priming conditions are shown for BMDMs transfected with LPS. (D) Additionally, a comparison between unprimed WT and Ifngr1−/− BMDMs treated with or without transfection reagent are shown. Bar graphs show the mean value +/− SEM along with individual data points pooled from independent experiments depicted with different shapes (A). Line graphs show the mean ± SD of three technical replicates (C and D). Data were pooled from two (A) independent experiments or are representative of two (B–D) independent experiments.

Statistical analysis performed using a two-way ANOVA and Tukey's multiple comparisons test; ∗∗∗∗ <0.0001; ∗∗∗ <0.002. See also Figure S4.