Additional file 1 of Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria
Creators: name: Kaczara, Patrycja
name: Czyzynska-Cichon, Izabela
name: Kus, Edyta
name: Kurpinska, Anna
name: Olkowicz, Mariola
name: Wojnar-Lason, Kamila
name: Pacia, Marta Z.
name: Lytvynenko, Olena
name: Baes, Myriam
name: Chlopicki, Stefan
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Virology

description: Additional file 1: Figure S1. Marker proteins in LSECs. Heat maps of the relative abundance (emPAI%) of marker proteins confirming LSEC purity. STAB2, Stabilin-2; OIT3, Oncoprotein-induced transcript 3; CLEC4G, C-type lectin domain family 4 member G; CLEC1B, C-type lectin domain family 1 member B; F8, Coagulation factor VIII; LYVE1, Lymphatic vessel endothelial hyaluronic acid receptor 1; FCGR2, Low affinity immunoglobulin gamma Fc region receptor II; EHD3, EH domain-containing protein 3; MRC1, Macrophage mannose receptor 1 (CD206); STAB1, Stabilin-1; PTPRB, Receptor-type tyrosine-protein phosphatase beta; AKAP12, A-kinase anchor protein 12; DAB2, Disabled homolog 2; ACP5, Tartrate-resistant acid phosphatase type 5; CD36, Platelet glycoprotein 4; FLT4, Vascular endothelial growth factor receptor 3; GPR182, G-protein coupled receptor 182; CDH5, Cadherin-5; MYCT1, Myc target protein 1; ADGRF5, Adhesion G protein-coupled receptor F5; ADGRL4, Adhesion G protein-coupled receptor L4; ENG, Endoglin; IGFBP7, Insulin-like growth factor-binding protein 7; PLP3, Phospholipid phosphatase 3. Data come from eight replicates. Figure S2. Energy metabolism enzymes in LSECs; proteomic analysis. (A-G; I-L) Heat maps of the relative abundance (emPAI%) of proteins identified in LSECs related to the glycolysis (A), TCA cycle and complex II of the ETC (B), complex I of the ETC (C), complex III of the ETC (D), complex IV of the ETC (E), complex V of the ETC (F), FA oxidation in mitochondria (G), peroxisomes and FA oxidation in peroxisomes (I), pyruvate metabolism (J), glutamine metabolism (K), and malate-aspartate shuttle (L). Data come from eight replicates. (H) Relative abundance (emPAI%) of 3-ketoacyl-CoA thiolase in LSECs; this component of the β-oxidation pathway shown in G is presented on a separate graph due to its abundance, which was far superior to the rest of the proteins identified in this pathway. Data come from eight replicates. Figure S3. Gene Ontology (GO)-enrichment proteomics analysis in LSECs. Gene enrichment analysis of proteins in LSECs. Functional enrichment analysis showing 20 the most significant categories of cellular components (A), biological processes (B) and molecular functions (C). Hierarchical clustering was performed with ShinyGO software 0.77. The charts provide information about GO fold enrichment (y-axis; pathways in order of the enrichment of FDR (false discovery rate)), significance (x-axis; FDR in log10), and number of proteins in each pathway. The color charts show the fold enrichment for each pathway. The size of dots corresponds to the number of genes assigned to each pathway. Data come from eight replicates for each type of cells. Figure S4. Assessment of oxidative phosphorylation and glycolysis in HAECs. A Heat map of the relative abundance (emPAI%) of selected proteins identified in HAECs related to the TCA cycle, ETC, FA oxidation in mitochondria (FAO), malate–aspartate shuttle (MAS), glutamine metabolism (G), peroxisomes and FA oxidation in peroxisomes (PX), and glycolysis. Data come from eight replicates. B Representative image of HAEC mitochondria. Mitochondria (green) were labelled with MitoTracker Green FM, and nuclei (blue) were labelled with Hoechst 33342. C Changes in ATP concentration in HAECs treated for 1 h with DMSO (as a vehicle), oligomycin (O; 1 µg/mL), or IAA (20 µM). Data are shown as means ± SEMs from three independent experiments (with three replicates per experiment). Significance was tested using one-way ANOVA (A and B); *, p &lt; 0.05. D The OCR (grey) and ECAR (blue) measured using the MST in HAECs; the mitochondrial function parameters were calculated from the kinetic data. Data are shown as means ± SEMs from three independent experiments (with 5–6 replicates per experiment). E The ECAR (blue) and OCR (grey) measured using the GST in HAECs; the glycolytic function parameters were calculated from the kinetic data. Data are shown as means ± SEMs from three independent experiments (with 5–6 replicates per experiment). Figure S5. Assessment of mitochondrial and glycolytic function in LSECs 2 h after cell isolation. A The OCR (grey) and ECAR (green) measured using the MST in LSECs 2 h after cell isolation. Data are shown as means ± SEMs from four biological replicates (with 5-6 technical replicates per biological replicate). B The ECAR (green) and OCR (grey) measured using the GST in LSECs 2 h after cell isolation. Data are shown as means ± SEMs from four biological replicates (with 5-6 technical replicates per biological replicate). Figure S6. Metabolic plasticity of LSECs. The OCR measured using the MFFT in LSECs in unbuffered DMEM supplemented with 5.5 mM glucose, 2 mM glutamine, and 1 mM pyruvate using the following reagents: UK-5099 (2 µM), BPTES (3 µM), and ETO (4 µM). Data are shown as means ± SEMs from four independent experiments (with 3–6 replicates per experiment). Figure S7. Effects of CPT1 inhibitors. (A and B) The OCR measured in LSECs in KHB supplemented with glucose (2.5 mM), untreated or treated with PER (5 µM; A) or OXF (1 mM; B), followed by DMSO or UK-5099 (10 µM) and CB-839 (5 µM), then FCCP (2 µM) and R/A (0.5/ 0.5 µM). Changes in basal respiration (90 min after PER or OXF addition) and maximal OCR were calculated from the kinetic data. Data are shown as means ± SEMs from three independent experiments (with 3-4 replicates per experiment). (C and D) The OCR measured in LSECs in KHB supplemented with glucose (2.5 mM), L-carnitine (50 µM) and PA-BSA (20 µM/3.3 µM), untreated or treated with PER (5 µM; A) or OXF (1 mM; B), followed by DMSO or UK-5099 (10 µM) and CB-839 (5 µM), then FCCP (5 µM) and R/A (1/1 µM). Changes in basal respiration (90 min after addition of PER or OXF) and maximal OCR were calculated from the kinetic data. Data are shown as means ± SEMs from three independent experiments (with 3-4 replicates per experiment). (E and F) The OCR measured in LSECs in KHB supplemented with glucose (2.5 mM), L-carnitine (50 µM) and BSA (3.3 µM), untreated or treated with PER (5 µM; A) or OXF (1 mM; B), followed by DMSO or UK-5099 (10 µM) and CB-839 (5 µM), then FCCP (5 µM) and R/A (1/1 µM). Changes in basal respiration (90 min after addition of PER or OXF) and maximal OCR were calculated from the kinetic data. Data are shown as means ± SEMs from three independent experiments (with 3-4 replicates per experiment). Figure S8. Carnitine palmitoyltranserase 1A. Relative CPT1A levels were quantified by Western blot analysis in protein lysates prepared from breast cancer MCF-7 cells, HAECs and LSECs. The relative levels of CPT1A were quantified by normalizing to the loading control. The protein concentration in each cell lysate was measured by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples containing 10 μg of total proteins were loaded into 7.5% SDS-PAGE gel, separated electrophoretically, and transferred onto PVDF membranes (Bio-Rad). Next, the membranes were blocked with 5% dry milk, and incubated overnight with the primary antibody (1:000) directed against the CPT1A (ab234111, Abcam) and with mouse IgG kappa binding protein (mIgGκ BP) conjugated to horseradish peroxidase (sc-516102, Santa Cruz Biotechnology), 1:5000 for 1 hour. Immunoreactive band detection was achieved by chemiluminescence (ChemiDocMP; Bio-Rad). Densitometric band analysis was performed using the ImageJ software. The total protein loaded onto the particular lane after transfer was used as the loading control using stain-free technology (Bio-Rad). Supplementary Table 1. Energy metabolism enzymes in LSECs; proteomic analysis. Supplementary Table 2. Energy metabolism enzymes in HAECs; proteomic analysis. Supplementary Table 3. Acylcarnitines levels in LSECs.