Supplementary Fig. S1.

(A–E). Inflammation promotes PAK1 activation. (A) Box plots compare PAK1 activation at the luminal intestinal surface and crypts in normal mucosa (n = 5), CD (n = 6), UC (n = 8), and also in CAC samples (n = 7). Tukey HSD; *p < 0.05, **p < 0.01, ***p < 0.001. (B) Immunofluorescence of p-PAK1 in TNFα-treated cells with DMSO (Con) or IPA-3 pretreatment. Upper panel shows p-PAK1; lower panel is p-PAK1 merged with DAPI staining. (C) WB of whole cell lysates (RIPA) in EV or WT-PAK1 overexpressing cells. TNFα activation of PAK1 was blocked upon IPA-3 pretreatment. Densitometry of relative p-PAK1 expression upon IPA-3 pretreatment, data are mean and SD from 2 independent experiments. (D) Inflammatory cytokines activate PAK1 in HCT116 cells. WB of RIPA lysates. Cells were left untreated (Con) or treated with IFNγ, IL-1β, or TNFα for 5–60 min and analyzed for PAK1 activation using p-PAK1 Thr 423 antibody. PAK1 activation increased from 5–60 min. Total PAK1 expression was also evaluated and α-tubulin was utilized as a loading control. (E) HCEC-1CT cells were co-cultured with PMNs in a 1:50 epithelial to neutrophil ratio and analyzed by WB for PAK1 expression. Total PAK1 expression increased from 30–60 min. Relative densitometry of PAK1 expression upon co-culture treatment. Data are mean and SD of 2 independent experiments.