Fig. 1: NSlit2 induces actin remodeling in macrophages in a ROBO1-dependent manner.

a Total RNA was isolated from murine brain and kidney, as well as from RAW264.7 cells and primary murine bone marrow-derived macrophages (BMDM). Robo1 and Robo2 mRNA expression were investigated by RT-PCR. Gapdh was used as a loading control. b A schematic representation of full-length human SLIT2 isoform 1 (accession number- NP_004778.1) protein, and recombinant NSlit2, CSlit2, and Slit2ΔD2 proteins used in this study. c RAW264.7 cells were incubated with vehicle (phosphate-buffered saline), NSlit2, CSlit2, or Slit2ΔD2 for 15 min at 37 °C and allowed to spread on poly-d-lysine-coated coverslips for 1 h. Cells were fixed, permeabilized, and incubated with AF-488-conjugated phalloidin (green) to label polymerized actin. Scale bar, 25 μm. d, e, g, h All data are presented as mean ± standard error of mean (SEM). Comparisons between the groups were made by one-way analysis of variance (ANOVA), followed by post hoc Tukey’s multiple comparison test. n = 50 cells per treatment group per experiment over three independent experiments. d Experiments were performed as in (c) and cell surface area was measured using Volocity 6.3 software. ****p < 0.0001, NSlit2 vs vehicle, CSlit2, or Slit2ΔD2. e Shape factor for cells in (c) using Volocity 6.3 software. ****p < 0.0001, NSlit2 vs vehicle, CSlit2, or Slit2ΔD2. f ROBO1 levels were knocked down in RAW264.7 cells using specific siRNA. Protein levels (band intensity) were quantified using ImageJ software, version 1.51v and normalized to corresponding β-actin levels. n = 3 independent experiments (Supplementary Fig. 1e). Comparison between the two groups was made by unpaired, two-tailed t-test. ***p = 0.0005, scrambled vs Robo1 siRNA. g ROBO1 protein levels were knocked down in RAW264.7 cells. After 72 h, experiments were performed as in (c). Cell surface area was measured as in (d). ****p < 0.0001 for the indicated comparisons and p = 0.9887, NSlit2 vs Slit2ΔD2 in ROBO1 knockdown conditions. (h) Shape factor for cells in (g) was determined as in (e). ****p < 0.0001 for the indicated comparisons and p = 0.4268, NSlit2 vs Slit2ΔD2 in ROBO1 knockdown conditions. Source data for (a, d, e, f–h) are provided as a Source Data file.