Fig. 6: NSlit2 attenuates protein uptake by macropinocytosis in cancer cells and restricts their growth in a ROBO1-dependent manner.

a–d All data are presented as mean ± SEM. Comparisons between the groups were made by Kruskal–Wallis ANOVA, followed by Dunn’s multiple comparisons test. PANC-1 and DLD-1 treatments are represented by black and gray symbols, respectively. a, d n = 8 independent replicates per treatment group per experiment over three independent experiments. a Oncogenic KRAS-expressing PANC-1 and DLD-1 cells were grown for 6 days in glutamine-free medium supplemented with high glutamine (Glut) (0.5 mM), low Glut (0.2 mM), 2% albumin. The macropinocytosis inhibitor, EIPA, was added, where indicated, for the last 2 days. Cell viability was measured on Day-6 using a MTT assay kit. Data are represented relative to the values obtained for 0.5 mM Glut supplementation. **p = 0.0015 and 0.0012, 0.5 mM vs 0.2 mM Glut for PANC-1 and DLD-1 cells, respectively; **p = 0.0013 and 0.0036, 0.2 mM Glut vs 0.2 mM Glut + 2% Alb for PANC-1 and DLD-1 cells, respectively; and *p = 0.0276 and 0.0360, 0.2 mM Glut + 2% Albumin vs 0.2 mM Glut + 2% Albumin + 25 µM EIPA for PANC-1 and DLD-1 cells, respectively. b, c n = 50 cells per treatment group per experiment over three independent experiments. b PANC-1 cells were incubated with vehicle, NSlit2, or Slit2ΔD2 for 15 min at 37 °C and macropinocytosis assays performed as described in Fig. 4a using Albumin-FITC in place of the TMR-labeled dextran. Mean fluorescence intensity (MFI) of the FITC channel per cell was measured using ImageJ software, version 1.51 v. ****p < 0.0001, NSlit2 vs vehicle, or Slit2ΔD2. c Experiments were performed as in (b) using DLD-1 cells instead of PANC-1 cells. p = 0.4469 and 0.2049, NSlit2 vs vehicle or Slit2ΔD2, respectively. (d) Cell viability assays were performed as described in (a) with addition of 0.2 mM glutamine, 2% albumin, NSlit2 and Slit2ΔD2 to the medium for 6 days, as indicated. **p = 0.0061 and >0.9999, 0.2 mM Glut + 2% Albumin vs 0.2 mM Glut + 2% albumin + NSlit2 for PANC-1 and DLD-1 cells, respectively. **p = 0.0048 and >0.9999, 0.2 mM Glut + 2% Albumin + NSlit2 vs 0.2 mM Glut + 2% Albumin + Slit2ΔD2 for PANC-1 and DLD-1 cells, respectively. Source data for (a–d) are provided as a Source Data file.