Fig. 4

ARL6IP1 localises to the tubular ER and mediates LD organisation in vitro. a Representative confocal images of ARL6IP1 immunostaining (red) with ER-tubule localising protein Sec61β (green) in U-2 OS cells. ARL6IP1 localises closely with Sec61β right out to the tips of peripheral ER tubules. b A single guide RNA (inset) was designed against the ARL6IP1 gene. The predicted cut site (3 bp upstream of the PAM) occurs immediately downstream of the nonsense mutation c.112C > T found to mutate an Arg residue to an early stop codon p.Arg38* in SPG61. c Lines generated from individual clones were screened by western blot to identify ARL6IP1 knockout lines with two independent ARL6IP1 knockout lines chosen for further experimentation. d Representative confocal images of LD540 stained LDs (green) in control and ARL6IP1 knockout cells. Average LD number per cell is significantly reduced by loss of ARL6IP1 (e) while average LD size is unaffected (f). Graphs represent averages from individual cells. n = 30 cells per genotype from 3 independent experiments. g Frequency distribution of LD sizes showing all LDs measured. Statistics consist of one-way ANOVA with Dunnett’s post-hoc tests