Supplementary Figure 6: Impairment of PtdIns4P disappearance by ORP1L KO clones and ORP1L variants.

a) TIDE analysis of a homozygous ORP1L CRISPR KO clonal RAW macrophage cell lines. b) Representative western blot showing expression of ORP1L in WT, control (cells stably expressing Cas9 and transduced with control sgRNA) and ORP1L KO RAW macrophage cell lines. c) Representative confocal micrographs of 3 independent replicates showing control or ORP1L KO RAW macrophages expressing GFP-2xP4M at the indicated times during phagosome maturation. Scale bars = 10 µm. d) PtdIns4P dynamics in maturing phagosomes of control cells (black bars), ORP1L KO clone “3” (white bars) and ORP1L KO clone “10” (stippled bars); means of ≥ 39 phagosomes per time point and per condition, from 3 experimental replicates ± SD; significance was determined by two-way ANOVA using Sidak correction, details are in the statistics source data supplementary file. e) Schematic representation of: (from top to bottom) WT ORP1L; ORP1L with a mutated FFAT motif (D478A) incapable of binding VAPA/B; ORP1L with a mutated lipid-binding pocket within the ORD (HH651/652AA) predicted to abolish PtdIns4P binding; ORPSAC1, in which the ORD was replaced with the phosphatase domain of Sac1 from S. cerevisiae. f) Schematic representation of the predicted behaviour of each ORP1L variant at ER-phagosome contacts. g-j) Representative maximum intensity projections of three independent replicates of RAW cells after 30 min of phagocytosis of SRBC expressing: g) GFP-ORP1L-FFAT* and mCh-VAPB; h) mCh-ORP1L-ORD* and GFP-VAPB; i) GFP-ORP1L-FFAT* and mCh-VAPA; and j) GFP-ORP1L-ORD* and mCh-VAPA. Insets show magnifications of each channel and merged image at bottom. Scale bars = 10 µm. k) Phagosomal 2xP4M (normalized to plasma membrane) in control cells, ORP1L KO cells (re-plotted from Fig. 5b) and for ORP1L KO cells expressing GFP-ORP1L-ORD*. Data are means ± SD of the indicated number of determinations from 3 independent experiments. Significance calculated using two-way ANOVA with Sidak correction, assuming statistical independence between groups, details are in the statistics source data supplementary file. l) Relative mRNA levels of control RAW cells treated with scrambled siRNA cells (black bar) or treated with siRNA against Orp1l, measured by qPCR; means ± SD of six independent experiments normalized individually to mRNA levels of paired cells treated with scrambled siRNA.