Additional file 1 of Nexilin promotes calcium-dependent endo-lysosomal fission required for retrograde transport

Supplementary Material 1: Figure S1. Nexilin does not affect lysosomal function or early endosome size. A Quantification of nexilin protein levels in PC3 cells transfected with control siRNA or six different siRNAs against nexilin from Western blotting, quantified using densitometry using ImageJ. The protein levels were normalized to tubulin, and plotted relative to the control. Data represents the mean ± s.d. from three independent experiments, two-tailed unpaired Student’s t-test was applied for statistical analysis. B U2OS cells were transfected with control siRNA, siRNAs targeting nexilin, or siRNA against Rab7a, and incubated with Magic Red for 1 hour before fixation and imaging. Scale bar: 10 µm. C Quantification of the average mean intensity of Magic Red per cell, relative to the control. Data represents the mean ± s.d. from 3 independent experiments; 2 experiments for siRab7a (n = 60 cells in total per condition, n = 40 cells in total for siRab7a), two-tailed, unpaired Student’s t-test was applied for statistical analysis. D U2OS cells transfected with control siRNA, siRNAs against nexilin or siRNA against Rab7a were lysed and subjected to Western blot analysis using antibodies against nexilin and Rab7a, and tubulin as loading control. E U2OS cells were transfected with siRNAs against nexilin or non-targeting control siRNA, and transiently transfected with GFP-Rab5 to label early endosomes before live cell imaging. Scale bar: 10 µm. F Quantification of early endosome size (µm²) per cell in U2OS cells transfected with control siRNA or siRNAs targeting nexilin. Data represents the mean ± s.d. for three independent experiments (n = 45 cells in total per condition), two-tailed unpaired Student’s t-test was applied for analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns non-significant