Fig 1: Inflammasome agonists cause Golgi retention of Hook2, but Hook2 and Fhip2a knockout does not affect inflammasome signalling in iBMDMs.(A-D) GolgiP volcano plots for (A) nigericin (20 μM, 30 min)-treated, (B) CL097 (75 μg/mL, 1 h)-treated, monensin (10 μM, 2 h)-treated, or (D) retro-2 (25 μM, 2 h)-treated versus untreated HEK293TEG cells. Dynein/dynactin and dynein activating adaptors are colored as shown. P values were calculated from the Student’s t-test (two sided) and adjusted for multiple hypothesis correction using the Benjamini–Hochberg approach (q-value) with MSstats. Data represent n=3 or 4 biological replicates. For the full TMTplex experimental setup, see Figure 5—Supporting Figures 1 and 4.(E) Inflammasome agonists collapse peripheral/cytosolic HOOK2 staining to the Golgi. HEK293T cells were treated with nigericin (20 μM, 30 min) or CL097 (75 μg/mL, 1 hr), fixed, and immunostained for HOOK2 and giantin (Golgi marker). Maximum intensity projections (8 μM over 29 steps) are representative of 2 independent experiments each with n > 7 fields of view. Scale bar = 10 μm.(F) Miseq genomic DNA results from Hook2 and Fhip2a (Fam160b1) knockout iBMDM clones. The gRNA reference sequence is colored in blue, gaps are indicated with dashes, and insertions are indicated with red letters.(G) Western blotting demonstrates that genomic knockout clones do not express either Hook2 or Fhip2a.(H-I) Hook2 and Fhip2a knockout does not alter (H) LDH or (I) IL-1β release from iBMDMs. Cells were treated with LPS (1 μg/mL, 4 hr) prior to the addition of nigericin (20 μM, 1 hr) or CL097 (75 μg/mL, 2 hr). Bar graphs represent mean ± standard deviation, n=3 biological replicates. ****p<0.0001 from a two-way ANOVA with Tukey’s post-test.(J) Hook2 and Fhip2a knockout does not alter ASC speck formation. The indicated primed (4 h LPS, 1 μg/mL) iBMDMs were treated with nigericin (0 or 20 μM, 30 min), fixed, immunostained, and imaged. Arrow heads point to ASC specks. Scale bar = 10 microns. Representative maximum intensity projection images (8 μM over 29 steps) from n > 7 fields of view.