Fig 2: Caveolin‐1 (Cav‐1) deficiency exacerbated silica‐induced inflammation. (A) Immunochemistry staining of CD68 and myeloperoxidase (MPO) in the lung sections from wild‐type (WT) and Cav‐1 −/− mice in the sham and silica 28 day groups. (B) The numbers of total cells in bronchoalveolar lavage fluid (BALF) from WT and Cav‐1 −/− mice in the sham and silica 28 day groups (n = 5). (C, D) Quantification of total macrophages (CD68‐positive cells) and neutrophils (MPO‐positive cells) in lung parenchyma of WT and Cav‐1 −/− mice in the sham and silica 28 day groups (n = 5). Relative mRNA levels of pro‐interleukin (IL)‐1β, IL‐6 and tumour necrosis factor‐ α (TNF‐α) in lung tissues from WT and Cav‐1 −/− mice in the sham and silica 28 day groups (n = 5). Western blot analysis of caveolin‐1 of WT and Cav‐1 −/− peritoneal‐derived macrophages (PDMs) after 24 h of silica exposure. Enzyme linked immunosorbent assay (ELISA) analysis of IL‐1β, TNF‐α and IL‐6 (n = 6) of WT and Cav‐1 −/− PDMs after 24 h of silica exposure. (H–I) PDMs were transfected with the Cav‐1 plasmid (Cav‐1 OE) and empty plasmid (Vehicle). Western blot analysis of Cav‐1 and ELISA analysis of IL‐1β, TNF‐α and IL‐6 (n = 3) of PDMs in Cav‐1 OE and vehicle groups after 24 h of silica treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 3: CAV1, CAV2, and PACSIN2 promote L. monocytogenes protrusion engulfment. (A) Infected A549 cells showing LmGFP (green) in a protrusion (arrow) or a vesicle (open triangle). Host membrane detected via β-catenin immunofluorescence (gray; magenta in merge). Scale bar = 2 μm. (B) Western blot analysis of A549 cell lysates after indicated RNAi treatments. (C, D) Percentage of L. monocytogenes in protrusions (C) or vesicles (D) in A549 cells after RNAi-mediated silencing of CAV1 or CAV2. (E) Infectious focus assay results after RNAi-mediated silencing of CAV1 (s2448), CAV2 (s2449), or PACSIN2 (s229822) expression in Caco-2 BBe1 cells. (F) Western blot analysis of Caco-2 BBe1 cell lysates after indicated RNAi treatments. (G, H) Percentage of L. monocytogenes in protrusions (G) or vesicles (H) in A549 cells after RNAi-silencing of PACSIN2. (I) Western blot analysis of A549 cell lysates after indicated RNAi treatments. Significance determined relative to the NT siRNA control using a Kruskal-Wallis ANOVA with Dunn’s multiple comparison test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, not significant.

Fig 4: Sphingolipid metabolism is a metabolic vulnerability in prostate cancer.a Viability curves for RM-9 and PC-3M cells following 48 h treatment with vehicle (ethanol), Eliglustat, PDMP or PPMP. b Unsupervised hierarchical clustering heatmaps illustrating differences in annotated lipid composition of RM-9 and PC-3M cells following 6 h challenge with either vehicle (ethanol), 128 µM Eliglustat, 64 µM PDMP, or 64 µM PPMP. Clustering was performed using Euclidean distance and Ward’s method. c Relative abundances ± StDev of ceramides and glycosphingolipids following 6 h treatment of RM-9 and PC-3M with either vehicle (ethanol) or 128 µM Eliglustat. n = 3 biologically independent replicates per experimental condition. Statistical significance was determined by two-sided student t-test comparing the aggregate intensities of individual lipid species corresponding to the respective lipid domain. d Immunoblots for LC3B-II following treatment of PC-3M cells with either vehicle or inhibitor as indicated in b. e Representative confocal microscopy images for PC-3M cells following 24 h pre-transfection with CellLight Lysosome-GFP (lysosomal associated membrane protein 1) and CellLight Mitochondria-RFP (leader sequence of E1 alpha pyruvate dehydrogenase) followed by acute (6 h) treatment with either vehicle or Eliglustat (128 µM). Scale bars indicate 50 µm. LUT intensity mapping was uniform for images of all treatment conditions. Immunoblots for LC3B and mitophagy markers Parkin and PINK1 following 6 h treatment with either vehicle (left) or eliglustat (128 µM, right). Targets were probed using two different membranes; asterisks (*/**) indicate corresponding control for each target. f Viability (MTS assay) ± StDev of PC-3M cells treated with 128 µM eliglustat after knockdown of CAV1 (n = 3 biologically independent replicates per experimental condition) or following pre-treatment with Cav-1 monoclonal blocking antibody (n = 12 and 4 biologically independent replicates per experimental condition for IgG and eliglustat treatment, respectively). Statistical significance was determined by two-sided student t-test. g Viability (MTS assay) ± StDev of LNCaP cells treated with 64 µM eliglustat following Cav-1 overexpression. n = 6 biologically independent replicates per experimental condition except for vehicle only treatment that consisted of n = 3 biologically independent replicates. Error bars given in all figures indicate statistical significance as determined by two-sided student t-test.

Fig 5: DJ1 oxidation—effects of Ang II and lipid raft disruptors. VSMCs treated with Ang II were subjected to discontinuous sucrose density gradient. (A) Representative immunoblots of total and irreversibly oxidised DJ1 in lipid rafts (LRs) vs. non-lipid rafts (NLRs). (B) VSMCs were stimulated with Ang II (100 nmol/L, 5 and 15 min) or treated with lipid-raft disruptors (10 mmol/L MCD or 50 μg/mL Nys). Representative immunoblots and quantification of total and irreversible oxidised DJ1. β-Actin was used as loading control. Data are means ± SEM from 7 experiments. Control was taken as 100% and data are presented as the percentage changes relative to control conditions. *P < 0.05 vs. control. Ang II angiotensin II, Ctl control, Cav-1 caveolin-1, LR lipid raft, NLR non-lipid raft, M marker, MCD methyl-b-cyclodextrin, Nys nystatin.