The role of RhoA and Cdc42 GTPase families in EphB2ΔC trans-endocytosis. Representative images (A) and quantification (B) showing the effect of RhoA subfamily (RhoA and RhoB) depletion on EphB2ΔC trans-endocytosis into ephrinB⁺ SKN cells. SKN cells were treated with siRNA for 72 h before 80-min co-culture with EphB2ΔC-GFP/Flag–positive cells. Cells were fixed without permeabilization and probed against Flag (surface EphB2ΔC, shown in red or yellow in the merge). Internalized vesicles appear as green puncta (total EphB2ΔC signal) within the vicinity of SKN nuclei (H2B channel, shown in blue). Image shown as maximum projection. Bars: 10 µm; (inset) 5 µm. Acquired images were analyzed using CellProfiler. (B) Results shown as mean ± SE (n = 4 independent experiments, >120 responder cells per condition per experiment, data normalized to median scramble value per experiment); ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, repeated measures one-way ANOVA with Dunnett’s post hoc test. (C) Quantification for the effect of RhoA subfamily depletion on EphB2ΔC trans-endocytosis into HeLa cells. HeLa cells overexpressing ephrinB1-mCherry were treated with siRNA for 48 h before 80 min co-culture with EphB2ΔC-GFP/Flag⁺ cells. Cells were fixed without permeabilization and probed against Flag (surface EphB2ΔC). Cells were manually scored for internal vesicles. Results shown as mean ± SE, n = 3 independent experiments, 19–36 responder cells per condition per experiment, statistics as in B. (D) Quantification showing the effect of RhoA subfamily depletion on EphB2-Fc endocytosis in ephrinB⁺ SKN cells, representative images shown in Fig. S4B. SKN cells were treated with indicated siRNAs before stimulation with fluorescently labeled, preclustered EphB2-Fc, fixed without permeabilization and stained against Fc (surface EphB2). Images were analyzed with CellProfiler^TM. Results shown as mean ± SE, n = 3 independent experiments, >463 cells per condition per experiment, data normalized to median scramble value per experiment; statistics as in B. (E) Quantification showing the effect of Cdc42 subfamily depletion on EphB2ΔC trans-endocytosis into ephrinB⁺ SKN cells. Experimental design and analysis as described in A and B (n = 3 independent experiments, >232 cells per condition per experiment, data normalized to median scramble value per experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.001, one-way ANOVA with Dunnett’s post hoc test.