Supplementary Figure 9: Analysis of human dermal fibroblasts obtained from a patient with a L1224R mutation in VPS15

a Sequencing traces of the A to C variant in VPS15 that encodes for a L1224R missense mutation in the homozygous proband. b Homology comparison of the L1224 residue in mice, xenopus, zebrafish, and drosophila reveals it is highly conserved. c Quantitation of relative mRNA expression of VPS15, VPS34 and BECLIN1 in dermal fibroblasts from the father, mother and proband. No significant differences were observed on the levels of VPS15 transcript (n = 3 flasks per individual; one-way ANOVA with a Tukey’s multiple comparison; father vs proband: P = 0.8928; mother vs proband: P = 0.9998), on the levels of VPS34 transcript (n = 3 flasks per individual; one-way ANOVA with a Tukey’s multiple comparison; father vs proband: P = 0.9429; mother vs proband: P = 0.2655) or on the levels of BECLIN1 transcript (n = 3 individual flasks; one-way ANOVA with a Tukey’s multiple comparison; father vs proband: P = 0.8377; mother vs proband: P = 0.1506). See Supplementary Table 1. These data indicate the that the reduction in VPS15 protein levels is due to post-transcriptional dysregulation. d Western blot analysis of LC3-I, and LC3-II on protein lysates prepared from patient and parent HDFs before and after lysosomal inhibition with Bafilomycin A1 (n = 3 flasks per individual). Cropped images are shown. Quantification e-g reveals a reduction in the ratio of LC3-II/LC3-I at 0hrs, but this is not statistically significant (one-way ANOVA with a Tukey’s multiple comparison, Bonferroni corrected; father vs proband: P>0.9999, mother vs proband: P>0.9999). Similarly when assessing autophagy flux (2hr-0hr) and formation at (4hr-2h) we observe a reduction in L1224R fibroblasts, however, this is not significant (one-way ANOVA with a Tukey’s multiple comparison, Bonferroni corrected; 2hr-0hr: father vs proband: P>0.9999, mother vs proband: P>0.9999; 4hr-2hr: father vs proband: P = 0.1836, mother vs proband: P = 0.1482). h Representative FACs plot for lysotracker experiments conducted on HDFs from the mother (shown in dark blue), father (shown in light blue), and proband (shown in red). The Y axis shows the cell count and the intensity of staining is shown on the X axis. i Quantification of the median intensity of lysotracker staining shows a mild but significant reduction in the proband in comparison to the father (n = 3 flasks per individual, one-way ANOVA with Tukey’s multiple comparison, father vs. proband P = 0.0466). Error bars indicate mean +/- standard error of the mean.