Fig 2: Wnt5a is involved in NLCs-induced survival and migration of CLL cells.A Cell viability analysis by trypan blue staining of CLL cells with or without NLCs treated by anti-Wnt5a or control mAb of irrelevant specificity for 24 h (n = 3). B Percentage of CLL cells migrating toward NLCs. CLL cells were treated by anti-Wnt5a or control mAb of irrelevant specificity for 4 h. Error bars denote standard deviation (n = 3). C Immunoblot analysis of phospho-ERK, total ERK, phospho-HS1, total HS1, phospho-p65, and total p65 in lysates of CLL cells with or without NLCs treated by anti-Wnt5a or control mAb of irrelevant specificity for 24 h. Dot figures represent ratios of densities (pERK/ERK, pHS1/HS1, and p-p65/p65).

Fig 3: Actin-dependent B-cell spreading during immune synapse formation is regulated by SNX5.(A) Representative epifluorescence images of Ctrl and SNX5-silenced B cells incubated with antigen-coated beads for different time points, stained for F-actin (red). Images are presented as z-projections of a stack. White circles indicate bead positions. The scale bar in bright field corresponds to 3 μm. (B) Quantification of the percentage of F-actin fluorescence around the bead. Values were normalized by the initial fluorescence (0 min). Two-way ANOVA with Sidak’s multiple comparison test was conducted for three independent experiments, with n > 25 cells. (C) Representative confocal images of Ctrl and SNX5-silenced B cells seeded onto antigen-coated coverslips for 30 and 60 min and stained for F-actin (red). Non-activated cells were seeded on poly-L-lysine. (D) Quantification of the spreading area of F-actin (μm2) from three independent experiments, with n > 30 cells. Two-way ANOVA with Sidak’s multiple comparison test was performed. * corresponds to the mean with respect to its control. & is the comparison of the mean between siCtrl and siSNX5-B. &&&& < 0.0001. (C, E) Quantification of actin foci in B cells from the experiment shown in (C) at 60 min of activation, for two independent experiments, with n > 25 cells. A t test was conducted. (F) Representative epifluorescence images of Ctrl and SNX5-silenced B cells incubated with antigen-coated beads for 10 and 30 min stained for F-actin (red) and B-cell receptor (green). The stack where the IS is located is shown. (G) Quantification of the B-cell receptor recruitment index to the center of the IS. Two-way ANOVA with Sidak’s multiple comparison test was conducted for two independent experiments, with n > 25 cells. (H) Representative confocal images of Ctrl and SNX5-silenced B cells seeded onto antigen-coated coverslips for 60 min and stained for p-HS1. The stack where the IS is located is shown. Dashed lines indicate cell boundaries (scale bars: 3 μm). (H, I) Quantification of fluorescence intensity of p-HS1 from images in (H). Two-way ANOVA with multiple comparison test under Tukey’s criteria was performed for two independent experiments. * corresponds to the mean with respect to its control. & is the comparison of the mean between siCtrl and siSNX5-B. &&&& < 0.0001.