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. 2023 Jan 9;30(2):589–604. doi: 10.1038/s41418-022-01106-9

Fig. 5. Intracellular Mobilization of GSDMD is Regulated by a MAP3K5:JNK Signaling Axis.

Fig. 5

A Coimmunostaining of pJNK and GSDMD in hMDM following extended priming and 2 h of 10 µM nigericin triggering. Yellow arrows indicate plasma-membrane localization. B Fluorescence lifetime (FLIM) of 488-pJNK in extended-primed hMDM triggered with 10 µM nigericin for 2 h. FLIM was quantitated as the reduction of Alexa 488-anti-pJNK fluorescence lifetime in the presence of Alexa 560-anti-GSDMD. C Extended-primed hMDM were treated with MAP3K5 and JNK inhibitors for one hour before triggering with 10 µM nigericin for 2 h. Cells were stained for pJNK and GSDMD to assess JNK activation and GSDMD localization. D Enumeration of hMDM pJNK mean cytosolic immunofluorescence following a 10 µM nigericin trigger ±30 µM M3K5i (selonsertib) or 30 µM JNKi (JNK-IN-8). E Quantitation of plasma-membrane localized GSDMD in hMDM treated as in (C). F CoIPs were performed on WT iBMDM or iBMDM stably expressing GSDMD-HA in naïve state (−) 100 nM KLA primed for 18 h (P) or additionally treated with 10 µM nigericin for 1 h (N) by immunoprecipitating HA and immunoblotting for indicated targets. G Protein interaction mapping was performed by co-expression in 293T17 cells with MAP3K5-FLAG fragments and either JNK2-HA or GSDMD-HA followed by IP of FLAG and immunoblotting of HA. Yellow arrows represent upshifted band migration. H SDS-resistant GSDMD super-shifting was performed by co-expressing MAP3K5, JNK2, and GSDMD in 293T17 cells in the presence or absence of MAP3K5 inhibitor [30 µM selonsertib]. A minimum of six confocal fields were acquired per sample condition per experiment (A–C) while quantitation represents five confocal (B, E) or 30 epifluorescent (D) fields in each of four replicates per group. B, D Error bars represent standard deviation from the mean. A, C Pearson’s correlation for pJNK/GSDMD colocalization was calculated by applying identical thresholding to experimental samples using Imaris. B, E Pair-wise two-tailed student’s T test of unequal variance; *p < 0.05, ***p < 0.001. D Two-way ANOVA followed by Tukey’s multiple comparison test; *p < 0.05, ****p < 0.0001. A–H Data shown are representative of at two (B, H) or three (A, C–H) experiments.