Additional file 5 of Nexilin promotes calcium-dependent endo-lysosomal fission required for retrograde transport

Supplementary Material 5: Figure S5. Nexilin depletion alters cholera toxin B retrograde transport to the Golgi and prevents LE/Lys fission. A U2OS cells were transfected with control siRNA, siRNAs against nexilin, and incubated with 4 mg/ml cholera toxin subunit B conjugated with AlexaFluor-555 (green) for 1 hour, then chased for 30 minutes before fixation and staining using a primary antibody against GM130 (magenta). White squares indicate magnified areas. Scale bar: 5 µm. B Quantification of colocalization of CTxB with GM130 using ImageJ to obtain Mander’s coefficient. Data represents mean ± s.d. from three independent experiments (n = 60 cells in total per condition). Two-tailed, unpaired Student’s t-test was applied for statistical analysis. C U2OS cells treated with control siRNA or siRNAs targeting nexilin were transiently transfected with GFP-Rab7b (green) before fixation and staining using rhodamine phalloidin (grey) and antibodies against Arp3 (magenta). White squares indicate magnified areas. Scale bar: 5 µm. D Quantification of the number of Arp3-positive actin clusters on Rab7b-positive endosomes per cell. Data represents mean ± s.d. from three independent experiments (n ≥ 41 cells in total per condition). Two-tailed, unpaired Student’s t-test was applied for statistical analysis. E U2OS cells were treated with control siRNA or siRNAs against nexilin, and transiently transfected with GFP-Rab7b before live cell imaging. White squares indicate the magnified area. Arrows point to fission tubules. Scale bar: 5 µm. F The graph represents the average number of Rab7b-positive fission tubules detected per hour per cell over 15 hours of live-cell imaging (n ≥ 9 cells in total per condition). Two-tailed, unpaired Student’s t-test was applied for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001