Fig. 2: NSlit2 activates RhoA and results in cytoskeletal rearrangement in macrophages.

(a, d, e, f) Data are presented as boxplots where middle line is the median; lower and upper hinges correspond to the first and third quartiles; whiskers represent minimum and maximum values. n = 50 cells per treatment group per experiment over three independent experiments. Comparisons between the groups were made by one-way ANOVA in (b, e, f) and two-way ANOVA in (a, d), followed by post hoc Tukey’s multiple comparison test. a Primary BMDM from wild-type (WT) or Rac1/2 double knockout (DKO) mice were cultured for 10 days, and cell spreading assays were performed as described in Fig. 1c. Cell surface area was measured as in Fig. 1d. ****p < 0.0001, WT vs DKO BMDM and p < 0.0001, NSlit2 vs vehicle, CSlit2, or Slit2ΔD2. b RAW264.7 macrophages were incubated with vehicle, NSlit2, CSlit2 or Slit2ΔD2 for 15 min at 37 °C and active and total RhoA levels were measured using a G-LISA assay. **p = 0.0063, 0.0093, and 0.0053 for NSlit2 vs vehicle, CSlit2, Slit2ΔD2, respectively. c RAW264.7 cells were incubated with the RhoA/B/C inhibitor, TAT-C3, for 4 h followed by vehicle, NSlit2, CSlit2, or Slit2ΔD2 treatment for 15 min and cell spreading was performed as described in Fig.1c. Scale bar, 20 μm. d Surface area for cells in (c) was measured as in Fig. 1d. ****p < 0.0001, vehicle vs TAT-C3 and p = 0.8877, 0.9470, and 0.9952 for NSlit2 vs vehicle, CSlit2, Slit2ΔD2 in TAT-C3-treated conditions, respectively. e, f Experiments were performed as in Fig. 1c but cells were first incubated with a formin inhibitor, SMIFH2, for 30 min. e Cell surface area was measured as in Fig. 1d. p = 0.9220, 0.8356, and 0.7215 for NSlit2 vs vehicle, CSlit2, Slit2ΔD2, respectively. f Shape factor was measured as in Fig. 1e. p = 0.7557, 0.6594, and 0.9950 for NSlit2 vs vehicle, CSlit2, Slit2ΔD2 respectively. Source data for (a, b, d, e, f) are provided as a Source Data file.