Fig 2: Stomatin interacted with CD36 and facilitated fatty acid uptake.a Immunoprecipitation (IP) assays revealed molecular interactions between stomatin and CD 36 in vitro. b Fatty acid uptake assays were performed to measure internalization of lipid ingredients by adipocyte-like cells from the extracellular environment. Fluorescently-labeled fatty acid (Bodipy-FL-C16) or fluorescent tag alone (Bodipy-FL) was added to the culture medium of adipocyte-like cells expressing exogenous stomatin (hSTOM), or control vector (Ctl). The degrees of Bodipy-FL-C16 and Bodipy-FL internalization were measured by intracellular accumulations of fluorescence over time. Mean ± s.e.m. for at n = 3 for Bodipy-FL-C16 n = 6 for Bodipy-FL uptake independent cells. c Immunofluorescence staining demonstrated that in cells treated with BSA, about half of the cell population contained stomatin-associated LDs. Treating cells with palmitic acid (PA) resulted in dissociation of STOM from LDs. Mean ± s.d. for three independent experiments. *P < 0.05 by two-sided unpaired t-test. Bar = 10 µm. d Surface biotinylation followed by Western blotting analyses of the biotinylated proteins were performed to quantify the surface portion (Surface) of a membrane protein, including stomatin, CD36, caveolin-1 (CAV1), or ATP1A1, relative to the total amount of individual proteins (Input). Actin proteins could not be labeled by surface biotinylation method and therefore contained no surface portion. Note increase of surface portion of stomatin after PA treatments (arrow) compared to the BSA control. In contrast, the surface portion of CD36, CAV1 remained unchanged after PA treatments. Mean ± s.d. for three independent experiments. *P < 0.05 by two-way ANOVA analysis. e, f Proximity ligation assays (PLA) were performed in 3T3-L1 adipocyte-like cells over-expressing exogenous human stomatin fused with GFP (hSTOM-GFP). Duolink® PLA reactions occurred when PLA probes for CD36 e or for CAV1 f were in closed proximity with hSTOM-GFP. Addition of connector oligos joined the PLA probes, resulting in in situ proximity ligation and formation of amplicon (arrowheads). e PA treatments promoted interactions between CD36 and stomatin on the plasma membrane, as evidence by the increased amplicons along cell surface compared with the control BSA treatments; removal of PA reversed such an increase. On the other hand, f PA treatments did not affect interactions between calveolin-1 and stomatin. Bar = 10 µm. Source data are provided as a Source data file.

Fig 3: Effect of mutations in the MHR1/2 domain and in the NUDT9H domain on activation of TRPM2 by ADPR and 2'-deoxy-ADPR. (a) Structure of TRPM2, visualization of PDB:6PUS [18] generated in UCSF chimera [34], MHR1/2 (blue) and NUDT9H (magenta) domains are highlighted. (b) Effect of mutations on the plasma membrane localisation of TRPM2. Transiently transfected HEK293 cells (as above, additionally, empty pIRES-EGFP vector control was used) were treated with a nonmembrane permeant biotinylating agent. Cells were harvested and total membrane proteins isolated (M). The biotinylated proteins were enriched using neutravidin agarose beads. After separation on a 4–15% SDS-PAGE proteins were transferred to a PVDF membrane and detected by chemoluminescence using primary antibodies against human TRPM2 (upper part, NB 500-241, Novus Biologicals) and human Na+/K+-ATPase ((lower part, #3010, Cell Signalling Technology) and an HRP-conjugated secondary antibody, both parts, Dianova #111-035-045) (c) HEK293 cells were either transiently transfected with expression vectors for human TRPM2 with mutations in either the NUDT9H domain (R1404Q) or in the MHR1/2 domain (R30/R358A) or the wild type channel. 24 h post-transfection cells were placed in a bath solution with NMDG and infused via the patch pipette with an intracellular solution buffered to a Ca2+ of 200 nM with EGTA and containing either ADPR, 2'-deoxy-ADPR or a combination of both. Max currents at +15 mV obtained from repetitive voltage ramps are shown on a log scale. Log-transformed data were tested against the respective control by one-way ANOVA followed by a post hoc t test with Bonferroni correction; the mean is indicated by a horizontal bar (**** p < 0.0001, ns not significant).

Fig 4: The loss of sacsin affects the localization of cell adhesion proteins(A) Western blot of cell surface protein purification, illustrated by the membrane protein ATP1A1, and the nuclear pore protein NUP98. After purification ATP1A1 is detectable only in conditions that were treated with biotin, and NUP98 is no longer detected, suggesting labeling specificity and enrichment of cell surface proteins.(B) Mass spectrometry of cell surface proteins in sacsin KO SH-SY5Y cells. Significance cutoffs: p < 0.05 and log2 fold change (f.c.) ±0.4, denoted by black outline. Proteins which pass these cutoffs, but were also detected in biotin negative controls, were not considered for downstream analysis (Table S1).(C) Western blot of NFASC in total lysate (left), and fractionated cytoplasmic or membrane fractions in WT and sacsin KO cells.(D) Levels of proteins detected in both cell surface and proteomic datasets. Proteins are colored by the disparity between these two datasets (f.c. surface and f.c. proteome), with red indicating more, and blue less membrane abundance relative to total protein levels. Black outlines are proteins with p < 0.05, log2 f.c. ± 0.4 in the surface dataset.(E) GO term analysis of proteins differentially localized in membrane of sacsin KO cells (p < 0.05, log2 f.c. ±0.4).(F and G) Confocal images for fibronectin and vimentin (F) and ER marker KDEL (G) in WT and sacsin KO HEK293 cells. Scale bars, 10 µm.(H) Disease enrichment analysis with Ingenuity Pathway Analysis of differentially localized surface proteins (p < 0.05, log2 f.c. ±0.4).