Fig 2: The SLIT2-ROBO1 axis triggers p38αMAPK.a Viability of SW-1990ROBO1-FL cells exposed to 30 nM rSLIT2 with or without the p38αMAPK-specific inhibitor VX-702 (5 μM) (n = 2 biological replicates, mean ± SEM., one-way repeated-measures ANOVA). The Y-axis represents OD values at 450 nm. b Viability of SW-1990CTRL and SW-1990ROBO1-FL cells treated with 30 nM rSLIT2 with or without the p38αMAPK-specific inhibitor VX-702 at various concentrations (0.2 μM, 1 μM, 5 μM, 25 μM, 125 μM; time point = D3; n = 2 biological replicates; mean ± SEM.; one-way Repeat-Measure ANOVA). ns. no significant difference, P > 0.05. The Y-axis represents OD values at 450 nm. c WB displaying the time-dependent rSLIT2-induced p38αMAPK phosphorylation in SW-1990Robo1-FL cells (n = 2 biological replicates). d WB showing the phosphorylation of p38αMAPK in PANC-1, MIA PaCa-2 or SW-1990 cell lines treated with 30 nM rSLIT2 (n = 2 biological replicates). e WB displaying the relationship of SLIT2 (30 nM), ROBO1 and MEK3/6 expression with phosphorylation of MEK3/6 or p38αMAPK (n = 2 biological replicates). f Representative ICC staining in 30 nM rSLIT2-treated SW-1990CTRL, SW-1990ΔROBO1 and SW-1990ROBO1-FL cells displaying the nuclear translocation of p38αMAPK (3 fields assessed per sample). P-p38MAPK, red; DAPI, blue. Scale bars: 50 μm (n = 2 biological replicates). g Representative ex vivo IHC-P staining of P-p38 showing rSLIT2-induced phosphorylation of p38αMAPK in cultured Panc02Robo1-FL cells from metastases in mouse livers (n = 6 samples per group, 3 fields assessed per sample). Scale bars, 100 μm. (h) Representative ex vivo IF staining of P-p38 and ROBO1 in KrasG12D/+/Trp53R172H/+/Pdx1-Cre (KPC) mouse model metastatic niches formed in livers with or without ROBO1 neutralizing antibody treatment. ROBO1, green; P-p38MAPK, red; DAPI, blue (n = 3 mice per group, 3 fields assessed per sample). Scale bar, 50 μm. I, j Lysate of the PANC-1 cell line endogenously immunoprecipitated with anti-ROBO1 and anti-p38α MAPK (i) or anti-ROBO1 and anti-MEK3/6 (j) antibodies and immunoblotted with the indicated antibodies (n = 2 biological replicates). k Lysate of rSLIT2 (30 nM)-induced SW-1990 cells immunoprecipitated with anti-ROBO1 antibody and immunoblotted with the indicated antibodies (n = 2 biological replicates). Source data are provided in the Source Data file.