Fig. 1: De novo NUS1 variants cause a spectrum of lysosomal defects in patient cells.

(a) Representative western blot for NgBR in wild type (WT) and patient fibroblasts and quantitation of NgBR levels relative to ß-actin (n = 3). Error = S.E.M. Statistical analysis was performed using a Dunnett’s test. (b) Quantitative polymerase chain reaction (PCR) analysis of NUS1 transcripts in WT and P1 fibroblasts (n = 3). The number of WT vs. variant alleles is shown. (c) Analysis of total dolichol and polyprenol levels in WT and patient fibroblasts. Analysis of the two patient lines (P1; n = 5 and P2; n = 3) were performed at different times using separate WT cells. Statistical analysis was performed using an unpaired Student’s t-test. (d) Representative western blots for ICAM1 and LAMP2 in WT and patient fibroblasts and quantitation of protein levels relative to HDAC1 (n = 3). Error = S.E.M. Statistical analysis was performed using a Dunnett’s test. (e) Representative western blot for NPC2 in WT and patient fibroblasts and quantitation of levels relative to ß-actin (n = 3). Error = S.E.M. Statistical analysis was performed using a Dunnett’s test. (f) Representative images of filipin-stained WT and patient fibroblasts and quantitation of pixel intensity in at least 40 different regions across 20 different cells. Error = S.E.M. Statistical analysis was performed using a Dunnett’s test. Scale bar = 10 µm. (g) Protein-normalized activity of acid-ß-glucosidase, ß-hexosaminidase, ß-glucuronidase, and ß-galactosidase in WT and patient fibroblasts. Arbitrary fluorescence units are plotted. Statistical analysis was performed using a Dunnett’s test. For all statistics *p < 0.05, **p < 0.01, ***p < 0.001. Red boxes indicate the additional correction (Dunnett’s test) was applied for comparison to a single control group.