Journal: The Journal of Cell Biology

Article Title: Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

doi: 10.1083/jcb.200905007

Figure Lengend Snippet: Osh4p has more than one membrane-binding surface. (A) The indicated mutations were introduced into Osh4p lacking endogenous cysteines (cys-less). The proteins were incubated with liposomes with the indicated lipid composition (N-MCC-PE contains a sulfhydryl-reactive headgroup) for 2 h at RT. The amount of protein pelleting with the membranes after washing (P) and the total amount of input protein (T) were determined by SDS-PAGE. The percentage of protein in P is shown ( n = 3). Black lines indicate that intervening lanes have been spliced out. (B) Single-cysteine mutants were allowed to react with a sulfhydryl-reactive NBD derivative. The ratio of the fluorescence of the proteins before (Fo) and after (F) the addition of liposomes (99:1 PS/PI(4,5)P2) was measured ( n = 6–8). (C, left) The structure of Osh4p (blue) bound to cholesterol (yellow) is shown. The flexible lid domain is shown in orange. (middle) A surface rendering of Osh4p, indicating the positively (blue) and negatively (red) charged surface. (right) The opposite side of the protein is shown. The positions of the residues changed to cysteine are indicated, and those that may interact with liposomes are shown in green. Error bars indicate mean ± SEM.

Article Snippet: The NEM-containing phospholipid N-MCC-PE was obtained from Avanti Polar Lipids, Inc. 10 µg Osh4p was incubated with sucrose-loaded liposomes containing PC/PE/MPB-PE/cholesterol/PI(4,5)P2 (59.5:29:1:10:0.5) for 2 h at room temperature.

Techniques: Binding Assay, Incubation, SDS Page, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

doi: 10.1083/jcb.200905007

Figure Lengend Snippet: Cholesterol extraction by Osh4p is regulated by PIPs in a second membrane. (A) 200 pmol Osh4p was incubated at 30°C with 500 µM 99:1 PC/[ 14 C]cholesterol liposomes together with 400 µM auxiliary liposomes containing 100% PC (red squares), 99.5:0.5 PC/PI(4,5)P 2 (blue triangles), or 99:1 PS/PI(4)P (gray triangles). All liposomes were sucrose filled. At the indicated times, the samples were placed on ice, and the liposomes were pelleted. The amount of [ 14 C]cholesterol in the supernatant was determined by scintillation counting. (B) 2 µM Osh4p was incubated at 30°C with 500 µM 99:1 PC/[ 14 C]cholesterol liposomes. After 15 min (arrow), auxiliary liposomes were added, and the amount of [ 14 C]cholesterol extracted was determined as in A ( n = 3). (C) Performed as in B except that auxiliary liposomes were added to a final concentration of 400 µM (1×), 800 µM (2×), or 1.6 mM (4×). The total incubation was 30 min at 30°C ( n = 4). Error bars indicate mean ± SEM.

Article Snippet: The NEM-containing phospholipid N-MCC-PE was obtained from Avanti Polar Lipids, Inc. 10 µg Osh4p was incubated with sucrose-loaded liposomes containing PC/PE/MPB-PE/cholesterol/PI(4,5)P2 (59.5:29:1:10:0.5) for 2 h at room temperature.

Techniques: Incubation, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activityin Vivo

doi: 10.1074/jbc.m604039200

Figure Lengend Snippet: FIGURE 1. oxPCCD36 and formation of oxPC-furan species. A, structures of phospholipids generated from oxidation of DHA-PC, PA-PC, and PL-PC that possess high affinity CD36 binding activity (oxPCCD36). The com- pounds generated from different precursors are similar except for the number of methylene groups on the truncated oxidized fatty acid esterified to the sn-2 positions of lyso-PC: n 2, DHA-PC; n 3, PA-PC; and n 7, PL-PC. B, suspected cyclization and dehydration steps through which oxPC-furan species are formed from their respective oxPCCD36 precursor.

Article Snippet: Materials 1,2-Ditridecanoyl-sn-glycero-3-phosphatidylcholine (DTPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PO-PC), PL-PC, PA-PC, andDHA-PCwere purchased fromAvanti Polar Lipids Inc. (Alabaster, AL).

Techniques: Generated, Binding Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activityin Vivo

doi: 10.1074/jbc.m604039200

Figure Lengend Snippet: FIGURE 5. oxPC-furans, in contrast to their oxPCCD36 precursors, do not bind to CD36. K562 cells trans- fected with plasmids containing intact human CD36 and GFP (bicistronic) were cultured overnight and then incubated with SUV (90 mol % oleic acid ester of 2-lyso-PC) containing 10 mol % of the indicated oxPCCD36 or oxPC-furan and tracer (1mol % of the lipophilic dye Di-I) at 4 °C for 60 min. After extensive washing to remove unbound SUV, CD36 expression and oxPC binding were analyzed simultaneously by two-color flow cytometry. Ashowsarepresentativeflowcytometrydotplot.B,themeanSUVbinding(Di-Ifluorescence)isplottedagainst the relative CD36 expression levels as monitored by the mean GFP fluorescence intensity for the various SUV examined. C and D, data shown represent oxPC binding for the single relative CD36 expression level (rectangle in B) in K562 cells transfected with either intact human CD36 and GFP (bicistronic) or GFP only (controls). E, binding studies with mouse peritoneal macrophages and Di-I-labeled SUV comprised of PO-PC only, or PO-PC carrier (90 mol %) and 10 mol % of either oxPC-furan(2) or HOHA-PC. The data shown represent the means S.D. for three independent experiments.

Article Snippet: Materials 1,2-Ditridecanoyl-sn-glycero-3-phosphatidylcholine (DTPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PO-PC), PL-PC, PA-PC, andDHA-PCwere purchased fromAvanti Polar Lipids Inc. (Alabaster, AL).

Techniques: Cell Culture, Incubation, Expressing, Binding Assay, Flow Cytometry, Fluorescence, Transfection, Labeling

Journal: Journal of Biological Chemistry

Article Title: Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activityin Vivo

doi: 10.1074/jbc.m604039200

Figure Lengend Snippet: FIGURE 9. Model of potential pathways impacting upon formation and decay of oxidized phospholipids that serve as high affinity ligands for the scavenger receptor CD36. CytoC, cytochrome c; RNS, reactive nitrogen species; MPO, myeloperoxidase; NOX, NAD(P)H oxidase; LPO, lipoxygenase; PAF-AH, platelet-activat- ing factor acetylhydrolase; OB-PL, ON-PL, and OV-PL, the 4-oxobutyric acid, 9-oxononanoic acid, and 5-oxova- leric acid ester of 2-lyso-PC; A-PL, G-PL, and S-PL, azeleic acid, glutaric acid, and succinic acid ester of 2-lyso-PC; oxPLCD36, oxidized phospholipids that bind to the scavenger receptor CD36; oxPC-furan, oxidized phospholip- ids with sn-2 acyl group that incorporates a terminal 2-furyl carbonyl.

Article Snippet: Materials 1,2-Ditridecanoyl-sn-glycero-3-phosphatidylcholine (DTPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PO-PC), PL-PC, PA-PC, andDHA-PCwere purchased fromAvanti Polar Lipids Inc. (Alabaster, AL).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: MIGA2 binds and transfers lipids between membranes. (A) Left: Native mass spectra of hMIGA2 long -6xhis purified from bacteria, showing exclusive presence of the dimeric protein. Each charge state is further accompanied by a series of up to four lipid bound peaks. Right: Expansion of the 20 + charge state of native MS for hMIGA2 long -6xhis dimer, showing up to four glycerophospholipids bound, with the average mass of 752 ± 5 Da. (B) Untargeted lipidomics of 3xFLAG-hMIGA2 long , purified from mammalian (Expi293) cells. At left: Distribution of lipid classes bound to hMIGA2 long . At right: Distribution of glycerophospholipids bound to hMIGA2 long as compared to their abundance in cells . (C) In the end-point transfer assay, hMIGA2 constructs are tethered between “heavy” acceptor liposomes (containing 0.75 M sucrose; lipid composition: 65% DOPC, 30% PE, and 5% PI[4,5]P 2 ) and light donor liposomes containing a single species of fluorescent lipid (containing no sucrose; lipid composition: 63% DOPC, 30% PE, 2% NBD- or Rh-lipid, 5% DGS-NTA). After lipid transfer (after 30 min), proteinase K was added to digest proteins, the light donor and heavy acceptor liposomes were separated by centrifugation, and the fluorescence increase of the heavy acceptor liposomes monitored. Transferred lipid was quantitated based on liposome standards incorporating 2, 1, 0.5, 0.25, or 0% of the appropriate fluorescent lipid. Both hMIGA2 long and hMIGA2 C transport NBD-PE acyl , -PS acyl , -PA acyl , and NBD-PC acyl ; NBD-PS head is transferred to a lesser extent, and Rh-PE head is not transferred, indicating that modification of the lipid headgroup can interfere with transport by MIGA2. In a similar experiment, we monitored transfer of unmodified PS using a PS-specific protein probe (C2-domain of lactadherin ) to assess PS transfer. Donor liposomes initially contained 5% PS. Donor and acceptor liposomes were separated by addition of EDTA and imidazole rather than protease, so as not to destroy the PS-probe. Supernatant (containing donor liposomes) and pellet (acceptor liposomes) fractions, in the presence and absence of MIGA2, were analyzed by SDS-PAGE to quantitate the PS-probe associated with each fraction. Transfer efficiency of acyl chain modified NBD-PS, as monitored by fluorescence, is comparable to that of natural PS, indicating that the acyl chain modification does not interfere with transfer by MIGA2. (D) In the FRET-based transfer assay, donor and acceptor liposomes (compositions indicated) were tethered together in the presence or absence MIGA2 linked to the donor liposomes. The donor liposomes initially contain Rh-PE head and NBD-lipids (-PS acyl , -PC acyl , -PA acyl , -PE acyl ), where FRET between the Rh and NBD initially reduces NBD fluorescence. As lipids are transferred from donor to acceptor liposomes, the Rh- and NBD-labeled lipids are diluted, resulting in reduced FRET and an increase in NBD fluorescence. hMIGA2 long can transport NBD-PS acyl , NBD-PA acyl , NBD-PE acyl , and NBD-PC acyl . A donor only control shows that the fluorescence increase was due to transfer of lipids between liposomes rather than solely lipid extraction by hMIGA2. After the transfer reaction was completed, dithionite was added to rule out the possibility of fusion between donor and acceptor liposomes, which would also result in a fluorescence increase . Each experiment was performed in triplicate. SDs are shown. NBD-PS head transfer is less efficient . (E) A similar FRET experiment was carried out to assess transfer from artificial LDs (rather than donor liposomes) to acceptor liposomes and shows that MIGA2 transfers NBD-PE acyl and NBD-PC acyl between the artificial LDs and liposomes. Positive stain transmission electron microscopy was used to assess the quality of the LD preparation as in .

Article Snippet: Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110).

Techniques: Purification, Construct, Centrifugation, Fluorescence, Modification, SDS Page, Labeling, Staining, Transmission Assay, Electron Microscopy

Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: Lipid binding and transfer by MIGA2. (A) 3xFLAG-hMIGA2 long was incubated with NBD-labeled lipids and examined by native PAGE. Phospholipids (NBD-PA acyl , NBD-PC acyl NBD-PS head , NBD-PE acyl ) visualized by their fluorescence, comigrated with protein, visualized by Coomassie blue staining. CH, cholesterol; SM, sphingomyelin. Because migration rates on native gels depend on the mass/charge ratio of the sample, and because MIGA2 dimers can bind multiple lipids at once, MIGA2 incubated with charged lipids migrates as multiple species and is smeared. (B) hMIGA2 long -6xhis was incubated with a 1:1 molar ratio of NBD-PC acyl and unlabeled lipid and examined by native PAGE. (C) Dithionite controls for the FRET transfer assay: After the transfer reaction was completed, dithionite was added to rule out the possibility of fusion between donor and acceptor liposomes, which would also result in a fluorescence increase. The fluorescence reduction after dithionite addition is the same for reactions containing hMIGA2 as those without, indicating that fusion has not occurred. Each experiment was performed in triplicate. SDs are shown. (D) FRET-based transfer experiment for NBD-PS head and the dithionite control. (E) Positive staining TEM micrographs of artificial LDs and liposomes: artificial LDs have a monolayer of phospholipids and TAG core, which are dark under positive staining; liposomes have bilayer membrane of phospholipids and buffer in the core, so that only the membrane is stained dark. Source data are available for this figure: .

Article Snippet: Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110).

Techniques: Binding Assay, Incubation, Labeling, Clear Native PAGE, Fluorescence, Staining, Migration

Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: MIGA2’s lipid transfer ability is essential for its function in mitochondria. (A) In two mutant forms of hMIGA2 (M1 and M2), hydrophobic residues in the lipid binding channel were altered to hydrophilic amino acids, indicated in yellow and cyan, respectively. In a third mutant (M3), in pink, a strictly conserved residue near the lipid headgroup was changed to asparagine (R357N in hMIGA2). The hMIGA2 C shown was modeled in AlphaFold2. (B) All three mutants lose ability to transfer lipids in the FRET-based assay with NBD-PE acyl . Each experiment was performed in triplicate. SDs are shown. (C) Knockout of MIGA2 in Hela cells was confirmed by Western blot against MIGA2. The upper non-specific band served as an internal loading control. The arrow indicates the band of MIGA2. (D) Mitochondria stained with MitoTracker Green in WT Hela cells are tubular, while in MIGA2 KO cells are fragmented. (E) Expression of WT MIGA2-DsRed and M1-, M2-, and M3-DsRed mutants in MIGA2 KO cells was compared by Western blot against MIGA2. The four MIGA2 constructs expressed at the same level when normalized to β-actin. (F) Expression of WT MIGA2-DsRed (PGK) in the KO cells rescued the mitochondria fragmentation morphology, whereas cells expressing either M1-, M2-, or M3-DsRed (PGK), the lipid transfer incompetent constructs, showed fragmented mitochondria. The dot expression pattern of MIGA2 constructs was consistent with previous studies . (G) Quantification of tubular and fragmented mitochondria in different cells ( n > 50). Statistical significance was calculated by Welch’s two-tailed unpaired t test. Results were indicated in the following manner: ∗ for P < 0.05, ∗∗ for P < 0.01, ∗∗∗∗ for P < 0.0001, where P < 0.05 is considered as significantly different. Graphs in the same panel are displayed with the same brightness and contrast settings. Scale bars in D and F, 5 μm. Source data are available for this figure: .

Article Snippet: Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110).

Techniques: Mutagenesis, Binding Assay, Knock-Out, Western Blot, Staining, Expressing, Construct, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: MIGA2 binds and transfers lipids between membranes. (A) Left: Native mass spectra of hMIGA2 long -6xhis purified from bacteria, showing exclusive presence of the dimeric protein. Each charge state is further accompanied by a series of up to four lipid bound peaks. Right: Expansion of the 20 + charge state of native MS for hMIGA2 long -6xhis dimer, showing up to four glycerophospholipids bound, with the average mass of 752 ± 5 Da. (B) Untargeted lipidomics of 3xFLAG-hMIGA2 long , purified from mammalian (Expi293) cells. At left: Distribution of lipid classes bound to hMIGA2 long . At right: Distribution of glycerophospholipids bound to hMIGA2 long as compared to their abundance in cells . (C) In the end-point transfer assay, hMIGA2 constructs are tethered between “heavy” acceptor liposomes (containing 0.75 M sucrose; lipid composition: 65% DOPC, 30% PE, and 5% PI[4,5]P 2 ) and light donor liposomes containing a single species of fluorescent lipid (containing no sucrose; lipid composition: 63% DOPC, 30% PE, 2% NBD- or Rh-lipid, 5% DGS-NTA). After lipid transfer (after 30 min), proteinase K was added to digest proteins, the light donor and heavy acceptor liposomes were separated by centrifugation, and the fluorescence increase of the heavy acceptor liposomes monitored. Transferred lipid was quantitated based on liposome standards incorporating 2, 1, 0.5, 0.25, or 0% of the appropriate fluorescent lipid. Both hMIGA2 long and hMIGA2 C transport NBD-PE acyl , -PS acyl , -PA acyl , and NBD-PC acyl ; NBD-PS head is transferred to a lesser extent, and Rh-PE head is not transferred, indicating that modification of the lipid headgroup can interfere with transport by MIGA2. In a similar experiment, we monitored transfer of unmodified PS using a PS-specific protein probe (C2-domain of lactadherin ) to assess PS transfer. Donor liposomes initially contained 5% PS. Donor and acceptor liposomes were separated by addition of EDTA and imidazole rather than protease, so as not to destroy the PS-probe. Supernatant (containing donor liposomes) and pellet (acceptor liposomes) fractions, in the presence and absence of MIGA2, were analyzed by SDS-PAGE to quantitate the PS-probe associated with each fraction. Transfer efficiency of acyl chain modified NBD-PS, as monitored by fluorescence, is comparable to that of natural PS, indicating that the acyl chain modification does not interfere with transfer by MIGA2. (D) In the FRET-based transfer assay, donor and acceptor liposomes (compositions indicated) were tethered together in the presence or absence MIGA2 linked to the donor liposomes. The donor liposomes initially contain Rh-PE head and NBD-lipids (-PS acyl , -PC acyl , -PA acyl , -PE acyl ), where FRET between the Rh and NBD initially reduces NBD fluorescence. As lipids are transferred from donor to acceptor liposomes, the Rh- and NBD-labeled lipids are diluted, resulting in reduced FRET and an increase in NBD fluorescence. hMIGA2 long can transport NBD-PS acyl , NBD-PA acyl , NBD-PE acyl , and NBD-PC acyl . A donor only control shows that the fluorescence increase was due to transfer of lipids between liposomes rather than solely lipid extraction by hMIGA2. After the transfer reaction was completed, dithionite was added to rule out the possibility of fusion between donor and acceptor liposomes, which would also result in a fluorescence increase . Each experiment was performed in triplicate. SDs are shown. NBD-PS head transfer is less efficient . (E) A similar FRET experiment was carried out to assess transfer from artificial LDs (rather than donor liposomes) to acceptor liposomes and shows that MIGA2 transfers NBD-PE acyl and NBD-PC acyl between the artificial LDs and liposomes. Positive stain transmission electron microscopy was used to assess the quality of the LD preparation as in .

Article Snippet: Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110).

Techniques: Purification, Construct, Centrifugation, Fluorescence, Modification, SDS Page, Labeling, Staining, Transmission Assay, Electron Microscopy

Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: Lipid binding and transfer by MIGA2. (A) 3xFLAG-hMIGA2 long was incubated with NBD-labeled lipids and examined by native PAGE. Phospholipids (NBD-PA acyl , NBD-PC acyl NBD-PS head , NBD-PE acyl ) visualized by their fluorescence, comigrated with protein, visualized by Coomassie blue staining. CH, cholesterol; SM, sphingomyelin. Because migration rates on native gels depend on the mass/charge ratio of the sample, and because MIGA2 dimers can bind multiple lipids at once, MIGA2 incubated with charged lipids migrates as multiple species and is smeared. (B) hMIGA2 long -6xhis was incubated with a 1:1 molar ratio of NBD-PC acyl and unlabeled lipid and examined by native PAGE. (C) Dithionite controls for the FRET transfer assay: After the transfer reaction was completed, dithionite was added to rule out the possibility of fusion between donor and acceptor liposomes, which would also result in a fluorescence increase. The fluorescence reduction after dithionite addition is the same for reactions containing hMIGA2 as those without, indicating that fusion has not occurred. Each experiment was performed in triplicate. SDs are shown. (D) FRET-based transfer experiment for NBD-PS head and the dithionite control. (E) Positive staining TEM micrographs of artificial LDs and liposomes: artificial LDs have a monolayer of phospholipids and TAG core, which are dark under positive staining; liposomes have bilayer membrane of phospholipids and buffer in the core, so that only the membrane is stained dark. Source data are available for this figure: .

Article Snippet: Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110).

Techniques: Binding Assay, Incubation, Labeling, Clear Native PAGE, Fluorescence, Staining, Migration

Journal: Current Biology

Article Title: Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

doi: 10.1016/j.cub.2020.05.083

Figure Lengend Snippet: Lysenin Specifically Binds Sphingomyelin (A) Constructs of Lysenin used in this study. Green, N-terminal domain; yellow, C-terminal domain. (B) Liposome flotation assay. Liposomes containing PC:Cholesterol (labeled PC) or Sphingomyelin:PC:Cholesterol (labeled SM) were mixed with recombinant Lysenin CTD -GFP purified from E. coli . After incubation, liposomes were floated and harvested. Proteins extracted from supernatant or liposomes were visualized by silver stain. I, input; S, supernatant; B, bound. (C) Cell-surface binding assay. Non-permeabilized HeLa cells were incubated with recombinant GFP, GFP-Galectin-8, Lysenin CTD -GFP, or Lysenin CTD,K185A -GFP, washed, and fixed. Binding was assessed by confocal microscopy and flow cytometry. Scale bar, 10 μm. Gray line, unstained; black line, stained with corresponding recombinant protein. (D) Effect of bSMase on Lysenin binding. Flow cytometry of untreated cells or cells pretreated with recombinant bacterial SMase followed by incubation with recombinant GFP-Galectin-8 or Lysenin CTD -GFP.

Article Snippet: Chicken Egg Sphingomyelin , Avanti Polar Lipids Inc. , Cat# 860061.

Techniques: Construct, Liposomes, Labeling, Recombinant, Purification, Incubation, Silver Staining, Binding Assay, Confocal Microscopy, Flow Cytometry, Staining

Journal: Current Biology

Article Title: Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

doi: 10.1016/j.cub.2020.05.083

Figure Lengend Snippet: Lysenin Is Recruited to Bacteria-Containing Vacuoles in a Sphingomyelin-Dependent Manner (A) Confocal micrographs of HeLa cells expressing GFP-Lysenin CTD or GFP-Lysenin W20A either uninfected or infected with mCherry-expressing S. Typhimurium 12023 and analyzed at 30 min post-infection (p.i.). Two micrographs of the same field of view are presented for HeLa cells expressing GFP-Lysenin W20A and infected with S. Typhimurium. The upper image was acquired with identical settings to the uninfected control; the lower image was acquired with a reduced 488 nm laser power. Scale bar, 10 μm. (B) Percentage of S. Typhimurium positive for Lysenin CTD or Lysenin W20A at 30 and 60 min p.i. Mean ± SEM of triplicate wells from three independent repeats. Automated image acquisition, automated quantification. n > 6,000 bacteria counted per well. (C) Confocal micrographs of HeLa cells expressing GFP-Lysenin W20A,K185A either uninfected or infected with mCherry-expressing S. Typhimurium and analyzed at 30 min post-infection (p.i.). Scale bar, 10 μm. (D) Percentage of S. Typhimurium positive for Lysenin W20A or Lysenin W20A,K185A at 30 and 60 min p.i. Mean ± SEM of triplicate wells from three independent repeats. Automated image acquisition, automated quantification. n > 6,000 bacteria counted per well. ∗∗∗∗ p < 0.0001, Student’s t test. (E) Confocal micrographs of HeLa cells expressing GFP-Lysenin W20A and mCherry-galectin-8 following treatment with different sterile damage-inducing reagents. White arrows indicate examples of Lysenin and galectin-8 co-localization. Scale bar, 10 μm (F) Confocal micrographs of HeLa cells expressing GFP-Lysenin W20A and mCherry-nSMase2 infected with S. Typhimurium and analyzed at 60 min p.i. Scale bar, 10 μm. (G) Percentage of S. Typhimurium positive for Lysenin W20A or galectin-8 in the presence or absence of ectopically expressed nSMase2. Mean ± SEM of triplicate wells from three independent repeats. Automated image acquisition, manual quantification. n > 700 bacteria counted per well. Ns, non-significant; ∗∗∗ p < 0.001, Student’s t test. (H) Percentage of S. Typhimurium wild-type (w/type) or Δ prgH + Inv positive for Lysenin W20A or galectin-8. Mean ± SEM of triplicate coverslips from three independent repeats. Quantification by eye using wide-field microscopy. n > 200 (w/type), n > 45 (Δ prgH + Inv ) bacteria counted per coverslip. ∗∗∗∗ p < 0.0001, Student’s t test. See also and and .

Article Snippet: Chicken Egg Sphingomyelin , Avanti Polar Lipids Inc. , Cat# 860061.

Techniques: Bacteria, Expressing, Infection, Control, Sterility, Microscopy

Journal: Current Biology

Article Title: Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

doi: 10.1016/j.cub.2020.05.083

Figure Lengend Snippet: Sphingomyelin Is Exposed on Vacuoles Containing Gram-Negative or Gram-Positive Bacteria and Exposure Occurs before Glycans (A) Confocal micrographs of HeLa cells expressing Lysenin W20A infected with Shigella flexneri M90T, Listeria monocytogenes EGD (BUG 600), Streptococcus pyogenes H293, or Enteropathogenic E. coli E2348/69 (EPEC). Confocal micrograph of THP-1 cells expressing Lysenin W20A infected with EPEC. HeLa scale bar, 10 μm; THP-1 scale bar, 5 μm. (B) Quantification of Lysenin recruitment to S. flexneri and L. monocytogenes . Mean ± SEM of triplicate coverslips from three independent repeats. Quantification by eye using wide-field microscopy. n > 100 bacteria counted per coverslip. (C) Percentage of S. Typhimurium positive for Lysenin W20A and/or galectin-8 at 30 and 60 min p.i. Mean ± SEM of triplicate wells from three independent repeats. Automated image acquisition, manual quantification. n > 700 bacteria counted per well. (D) Structured illumination micrographs of HeLa cells expressing mCherry-Lysenin W20A and YFP-galectin-8 infected with blue fluorescent protein (BFP)-expressing S. Typhimurium and fixed at 60 min p.i.. Scale bar, 1 μm. (E) Structured illumination micrographs of HeLa cells expressing mCherry-Lysenin W20A infected with BFP-expressing S. Typhimurium and stained for galectin-8 and NDP52 at 60 min p.i.. Scale bar, 1 μm. (F) Quantification of the time interval between Lysenin and galectin-8 recruitment to S. Typhimurium or S. flexneri visualized by live-cell imaging. n > 107 S. Typhimurium and n > 45 S. flexneri events were analyzed. Mean ± SD indicated. ∗∗∗∗ p < 0.0001, Student’s t test. (G) Tracking of a Salmonella -containing vacuole visualized by live-cell imaging. Graph indicates mean 488 nm (green) and 561 nm (red) fluorescence intensity changes around the bottom bacterium in . Time indicated is from the start of the track rather than bacterial entry into the cell. Graph shown is a representative example. See also .

Article Snippet: Chicken Egg Sphingomyelin , Avanti Polar Lipids Inc. , Cat# 860061.

Techniques: Bacteria, Expressing, Infection, Microscopy, Staining, Live Cell Imaging, Fluorescence

Journal: Current Biology

Article Title: Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

doi: 10.1016/j.cub.2020.05.083

Figure Lengend Snippet: Sphingomyelin Is Exposed on the Cytosolic Leaflet of BCVs before Cytosolic Entry of the Bacterium (A) Selected frames from super resolution live-cell imaging of HeLa cells expressing mCherry-Lysenin W20A and YFP-galectin-8 infected with BFP-expressing S. Typhimurium shown in . White arrow indicates appearance of a break in the SCV membrane. Scale bar, 5 μm. (B) Selected frames from live-cell imaging of HeLa cells expressing mCherry-Lysenin W20A and CFP-galectin-8 infected with GFP-expressing S. flexneri shown in . Video and corresponding stills were false colored to aid visualization: green, Lysenin W20A ; red, galectin-8; blue, S. flexneri. White arrow indicates appearance of a break in the SfCV membrane. Scale bar, 2 μm. (C and D) Correlative fluorescence and electron microscopy of HeLa cells expressing mCherry-Lysenin W20A and YFP-galectin-8 infected with BFP-expressing S. Typhimurium 30–40 min post-infection. Left panel: fluorescence images of ∼300 nm sections through resin-embedded cells, merge of red (mCherry signal) and green (YFP signal) channel. White square corresponds to area shown in second panel, imaged by electron tomography. Second panel: virtual slice through electron tomogram, showing SCV identified by fluorescence microscopy. White square corresponds to the area magnified in the third panel. Yellow arrows indicate ruptures of the SCV membrane. Right panel shows 3D segmentation models of the SCV membrane (yellow) and the surface of the bacterium (green). The background is a different virtual slice of the tomogram shown in the second panel. Scale bar, 1 μm (left panel), 100 nm (second and right panel), and 50 nm (third panel). See also , , , and .

Article Snippet: Chicken Egg Sphingomyelin , Avanti Polar Lipids Inc. , Cat# 860061.

Techniques: Live Cell Imaging, Expressing, Infection, Membrane, Fluorescence, Electron Microscopy, Tomography, Microscopy

Journal: bioRxiv

Article Title: Immobilisation of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

doi: 10.1101/2023.01.10.523433

Figure Lengend Snippet: illustrates the structural formulas of the phospholipids labelled with Cy5 PE (A), CL (B), and PC (C). Cutting points of the phospholipases applied (phospholipase A2 and phospholipase C) marked on the example of phosphatidylethanolamine Cy5. Phospholipase A2 cuts the ester bond to the second fatty acid chain at the sn-2 position ( ; ). Phospholipase C catalyses the hydrolysis of the phosphodiester bond to the glycerol backbone .

Article Snippet: cardiolipin - Cy5, phos-phatidylethanolamine - Cy5, phosphatidylcholine - Cy5, were purchased from Avanti Polar Lipids, INC, Birmingham, Alabama.

Techniques:

Journal: bioRxiv

Article Title: Immobilisation of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

doi: 10.1101/2023.01.10.523433

Figure Lengend Snippet: Hydrophobic microbead populations MB1 - MB4 were successfully coupled with 20 μg/μL of phospholipids CL-Cy5, PE-Cy5 and PC-Cy5. The coupling of PE-Cy5 reaches the highest fluorescence signal and CL-Cy5 represents the coupling with the lowest fluorescence signal. All measured signals differ significantly from each other (significances given as * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: cardiolipin - Cy5, phos-phatidylethanolamine - Cy5, phosphatidylcholine - Cy5, were purchased from Avanti Polar Lipids, INC, Birmingham, Alabama.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Immobilisation of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

doi: 10.1101/2023.01.10.523433

Figure Lengend Snippet: Hydrophobic microbead populations MB1, MB2, MB3 and MB4 were coupled with different concentration of CL-Cy5 (0.1 μg/μL to 200 μg/μL) to find out the optimal loading density of the fluorescently labelled phospholipid. The highest fluorescence value is reached at a concentration of 100 μg/μL. From a concentration of 200 μg/μL, the fluorescence values decrease again.

Article Snippet: cardiolipin - Cy5, phos-phatidylethanolamine - Cy5, phosphatidylcholine - Cy5, were purchased from Avanti Polar Lipids, INC, Birmingham, Alabama.

Techniques: Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Immobilisation of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

doi: 10.1101/2023.01.10.523433

Figure Lengend Snippet: Verification of the directional binding of phospholipids using the example of the fluorescently labelled phospholipid PC-Cy5 to the hydrophobic surface of the microbead populations MB1 (A), MB2 (B), MB3 (C) and MB4 (D) by the phospholipases A2 and C. Visible is the initial fluorescence and the associated Cy5 corona on the microbead (E) and the decrease in fluorescence due to hydrolysis and the associated cleavage of the fluorescently labelled head (F). The images were taken using a confocal microscope.

Article Snippet: cardiolipin - Cy5, phos-phatidylethanolamine - Cy5, phosphatidylcholine - Cy5, were purchased from Avanti Polar Lipids, INC, Birmingham, Alabama.

Techniques: Binding Assay, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Immobilisation of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

doi: 10.1101/2023.01.10.523433

Figure Lengend Snippet: Fluorescently labelled phospholipids CL-Cy5, PE-Cy5 and PC-Cy5 are bound to the surface of hydrophilic microbead populations (MB5). When coupling the phospholipids to hydrophilic microbeads PE-Cy5 shows the highest ligand fluorescence, whereas CL-Cy5 shows the lowest ligand fluorescence upon coupling (significances given as * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: cardiolipin - Cy5, phos-phatidylethanolamine - Cy5, phosphatidylcholine - Cy5, were purchased from Avanti Polar Lipids, INC, Birmingham, Alabama.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Immobilisation of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

doi: 10.1101/2023.01.10.523433

Figure Lengend Snippet: Hydrophilic microbeads coupled with fluorescently labelled phospholipids CL-Cy5, PE-Cy5, PC-Cy5 (A) were incubated with phospholipase PLC to show that fluorescence is not reduced at the microbeads and thus it is mandatory to use a hydrophobic microbead surface to bind the fluorescently labelled phospholipids directionally, with the hydrophobic part. The images in 7 B without enzyme and with enzyme also show no decrease in fluorescence. The images were taken using a confocal microscope.

Article Snippet: cardiolipin - Cy5, phos-phatidylethanolamine - Cy5, phosphatidylcholine - Cy5, were purchased from Avanti Polar Lipids, INC, Birmingham, Alabama.

Techniques: Incubation, Fluorescence, Microscopy

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Engineering a protein Z-dependent protease inhibitor (ZPI) mutant as a novel antagonist of ZPI anticoagulant function for hemophilia treatment

doi: 10.1111/jth.14610

Figure Lengend Snippet: A, Crystal structure of the ZPI (cyan)-PZ (green) complex (PDB:3H5C). The mutated amino acids are in blue sphere. ZPI residues that bind PZ are in red stick. B, Competitive binding of ZPI-2A, WT ZPI and PZAb with NBD-ZPI complexed with PZ. ZPI-2A (open circles), WT ZPI (closed circles) or PZAb (triangles) were titrated into 50nM K239C-NBD ZPI/63nM PZ complex in I 0.15 Tris buffer pH 7.4, 1.5mM CaCl2, 1 mg/ml BSA at 25°C and the decrease in NBD fluorescence due to displacement of PZ from the labeled ZPI monitored. Solid lines are computer fits of titrations by the competitive binding equation which yielded the reported KDs in the text for competitor binding. C, ZPI-2A blocks WT ZPI/PZ inhibition of membrane-associated FXa. Reactions contained ~20nM WT ZPI/PZ complex, 25μM PBE SUVs, 5mM Ca2+, 0.5nM FXa, and the indicated concentrations of ZPI-2A in I 0.15 Tris buffer, pH 7.4, 25°C. The solid line is a fit by the competitive binding equation. D, ZPI-2A blocks WT ZPI/PZ inhibition of prothrombinase-bound FXa. Reactions contained ~50 nM WT ZPI/PZ complex, 15nM FVa, 0.5nM FXa, 25μM PBE SUVs, 5mM Ca2+ and the indicated concentrations of ZPI-2A in I 0.15 Tris buffer, pH 7.4 at 25°C. The solid line is a fit by the competitive binding equation. E, ZPI-2A or the PZAb reverses WT ZPI-PZ inhibition of prothrombinase activation of prothrombin. Reactions contained ~50nM WT ZPI-PZ complex, 15nM FVa, 0.03nM FXa, 25μM PBE SUVs, 5mM Ca2+, 1.4μM prothrombin and the indicated concentrations of ZPI-2A or 200μg/ml PZAb in I 0.15 Tris buffer, pH 7.4 at 37°C. F, ZPI-2A reverses WT ZPI-PZ inhibition of thrombin generation in ZPI/PZ-deficient plasma. Reactions were performed in ZPI/PZ-deficient plasma (100μl) primed with plasma levels of WT ZPI and PZ in the absence and presence of the indicated final concentrations of ZPI-2A in I 0.15 Tris buffer, pH 7.4 at 37°C. Coagulation was activated by adding SUVs (20% PS/20% PE/60%PC), Ca2+, CTI (to block contact activation) and TF to a total volume of 150μl. Final concentrations were 1.5-fold dilution citrated plasma, 35nM ZPI, 35nM PZ, 25μM SUVs, 16mM Ca2+, 50μg/ml CTI, 2pM TF, 0–500nM ZPI-2A. G, ZPI-2A increases thrombin generation in FVIII-deficient hemophilia plasma. Reactions were performed in FVIII deficient plasma (100μl) in the absence and presence of the indicated concentrations of ZPI-2A(0–100nM), PZAb (0–200μg/ml) or control sheep IgG (Sigma-Aldrich, St. Louis, MO) in I 0.15 Tris buffer, pH 7.4 at 37°C. Coagulation was activated by adding SUVs(20% PS/20% PE/60%PC), Ca2+, CTI and TF to a total volume of 150μl. Final concentrations were 1.5-fold dilution citrated plasma, 25μM SUVs, 16mM Ca2+, 50μg/ml CTI, 2pM TF. All data represent the average of 3–4 independent measurement ± SD for figure 1B–1G.

Article Snippet: Small unilamellar phospholipid vesicles (SUVs) made from porcine brain extract(PBE) (Polar extract, 18.5% phosphatidylserine (PS)/33% phosphatidylethanolamine(PE)/13% phosphatidylcholine (PC)) or from 20% synthetic dioleoyl PS and 20%PE/60%PC from bovine heart (Avanti, Alabaster, AL) were prepared as described[ 8 , 13 , 14 ].

Techniques: Binding Assay, Fluorescence, Labeling, Inhibition, Activation Assay, Coagulation, Blocking Assay

Journal: Biochimica et biophysica acta

Article Title: Interaction of Ceramides and Tear Lipocalin

doi: 10.1016/j.bbalip.2018.01.004

Figure Lengend Snippet: Molar Extinction Coefficients in Solvents of Tested Compounds

Article Snippet: C6-NBD ceramide (N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine) and C12-NBD ceramide (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine) were obtained from Avanti Polar Lipids (Alabaster, AL).

Techniques: Solvent

Journal: Biochimica et biophysica acta

Article Title: Interaction of Ceramides and Tear Lipocalin

doi: 10.1016/j.bbalip.2018.01.004

Figure Lengend Snippet: Binding Parameters Calculated from Fitting of Ligand Binding to Tear Lipocalin

Article Snippet: C6-NBD ceramide (N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine) and C12-NBD ceramide (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine) were obtained from Avanti Polar Lipids (Alabaster, AL).

Techniques: Binding Assay, Ligand Binding Assay, Fluorescence

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation

doi: 10.1161/ATVBAHA.125.322448

Figure Lengend Snippet: LXR (liver X receptor) inhibition leads to cholesterol and oxysterol accumulation and cholesterol ester remodeling in macrophages. A , Relative mRNA levels of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1) in vehicle-treated human monocyte-derived macrophages (hMDMs; hMDM dimethylsulfoxyde [DMSO]), atherosclerotic plaque extract–treated hMDMs (hMDM plaque), and atherosclerotic plaque extract–treated and LXR inhibitor–treated hMDMs (hMDM plaque GSK2033 [GSK]) were assessed by RT-qPCR (quantitative reverse transcription polymerase chain reaction). Each dot represents the average response of an individual macrophage donor (nhMDM=5) treated with 3 to 6 different plaques. Bars represent mean with SD. * P <0.05, **** P <0.0001, 2-way ANOVA was used with an uncorrected Fisher LSD in post hoc analysis; normality was confirmed with the Shapiro-Wilk normality test. B , Total cholesterol and cholesteryl esters in hMDMs treated with atherosclerotic plaque extracts (hMDM plaque) or atherosclerotic plaque extracts and LXR inhibitor (hMDM plaque GSK) were assessed by GC-MS and LC-MS/MS, respectively. Free cholesterol was calculated by subtracting cholesteryl esters from total cholesterol. Each dot represents the response of an individual macrophage donor (nhMDM=8) treated with a mix of 6 different plaques; * P <0.05, Wilcoxon test was performed. C , Total cholesterol concentration in the supernatant of hMDMs conditioned with atherosclerotic plaque extracts, treated with or without the LXR inhibitor, was assessed by GC-MS. Each dot represents the response of a macrophage donor (nhMDM=9) treated with a mix of 6 different plaques. ** P <0.01, Wilcoxon test was performed. D , Confocal microscopy images showing staining of nuclei with DAPI (4′6-diamidino-2-phenylindole; blue) and membrane cholesterol with GFP-D4 probe (green fluorescent protein D4; green) in hMDMs following treatment with atherosclerotic plaque extracts and vehicle (plaque+DMSO) or atherosclerotic plaque extracts and LXR inhibitor (plaque+GSK). E , Western Blot analysis of ApoE from plaque extracts and vehicle-treated hMDMs (DMSO+plaque) and plaque extracts and LXR inhibitor–treated hMDM (GSK+plaque) supernatant form 1 hMDM donor treated with different plaque extracts. Ponceau S is used as a loading control. Graphic represents the comparison of ApoE expression relative to Ponceau S coloration between the 2 conditions. Each dot represents the response of the same hMDM donor treated with 5 different plaques. * P <0.05. Variables passed the Shapiro-Wilk normality test and were analyzed with a paired t test. F , Bubble plot illustrating the relative enrichment of cholesteryl ester species in atherosclerotic plaque extract–conditioned hMDMs treated with or without the LXR inhibitor. The x axis represents the log2 fold change between conditions with and without the LXR inhibitor, while the y axis represents −log10 of P values. Bubble size indicates the relative proportion of cholesteryl ester species in the LXR inhibitor condition. Red bubbles indicate statistically significant variations between the 2 conditions, as determined by the Wilcoxon test. Each dot represents the response of an individual macrophage donor (nhMDM=8) treated with a mix of 6 plaques.

Article Snippet: On day 6, hMDMs were treated for 48 hours with 1 of the following treatments: whole atherosclerotic plaque extracts (1% v/v in cell culture medium), 10 μmol/L 27-hydroxycholesterol (27OHC; Avanti Polar Lipids; Sigma; France; catalog No. 20380-11-4), or 50 μg/mL oxidized low-density lipoprotein, with or without LXR antagonist: 1 μmol/L of GSK (Sigma-Aldrich, St. Louis, MO; catalog No. 1221277-90-2) or 10 μmol/L of SR9238 (MedChemExpress, Monmouth Junction, NJ; catalog No. HY-101442).

Techniques: Inhibition, Binding Assay, Derivative Assay, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Confocal Microscopy, Staining, Membrane, Western Blot, Control, Comparison, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation

doi: 10.1161/ATVBAHA.125.322448

Figure Lengend Snippet: LXR (liver X receptor) inhibition induces oxysterol accumulation and modulates enrichment of 25-hydroxycholesterol in both intracellular and supernatant fraction. A through E , Oxysterol levels (7α-OH-cholesterol [ A ], 7β-OH-cholesterol [ B ], 7-ketocholesterol [ C ], 25-OH-cholesterol [ D ], and 27-OH-cholesterol [ E ]) in human monocyte-derived macrophages (hMDMs) treated with atherosclerotic plaque extracts alone (hMDM plaque) or with the addition of an LXR inhibitor (hMDM plaque GSK2033 [GSK]) were assessed using GC-MS (gas chromatography-mass spectometry). Each dot represents the response of an individual macrophage donor (n hMDM =8) treated with a mix of 6 different plaques. * P <0.05 Wilcoxon test. F through H , Concentrations of oxysterols (7α-OH-cholesterol [ G ], 7β-OH-cholesterol [ H ], and 25-OH-cholesterol [ I ]) in the supernatant of hMDMs conditioned with atherosclerotic plaque extracts, with or without LXR inhibitor treatment, were assessed using GC-MS. Each dot represents the response of an individual macrophage donor (n hMDM =9) treated with a mix of 6 different plaques. * P <0.05; Wilcoxon test. I , Bubble plot depicting oxysterol species normalized by the total cholesterol concentration in the supernatant of hMDMs conditioned with atherosclerotic plaque extracts, with or without LXR inhibitor treatment. The x axis represents the log2 fold change between conditions with and without the LXR inhibitor, while the y axis represents −log10 of P values. Bubble size indicates the relative levels of oxysterol species in the LXR inhibitor condition. Red bubbles indicate statistically significant variations between the 2 conditions, as determined by the Wilcoxon test. Each dot represents the response of an individual macrophage donor (n hMDM =9) treated with a mix of 6 different plaques.

Article Snippet: On day 6, hMDMs were treated for 48 hours with 1 of the following treatments: whole atherosclerotic plaque extracts (1% v/v in cell culture medium), 10 μmol/L 27-hydroxycholesterol (27OHC; Avanti Polar Lipids; Sigma; France; catalog No. 20380-11-4), or 50 μg/mL oxidized low-density lipoprotein, with or without LXR antagonist: 1 μmol/L of GSK (Sigma-Aldrich, St. Louis, MO; catalog No. 1221277-90-2) or 10 μmol/L of SR9238 (MedChemExpress, Monmouth Junction, NJ; catalog No. HY-101442).

Techniques: Inhibition, Derivative Assay, Gas Chromatography-Mass Spectrometry, Gas Chromatography, Concentration Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation

doi: 10.1161/ATVBAHA.125.322448

Figure Lengend Snippet: Cytokine response following LXR (liver X receptor) modulation. A , Volcano plot depicting differentially expressed genes identified from RNA-sequencing data comparing untreated human monocyte-derived macrophages (hMDMs) to plaque-exposed hMDMs. Red dots represent genes with a –log( P value) >1. Blue dots represent genes with expression levels below this threshold. Three hMDM donors were treated with 3 different plaque extracts. A Wald test was performed to generate P values and log2-fold changes. B , Volcano plot illustrating differentially expressed genes identified from RNA-sequencing data comparing plaque-exposed hMDMs to those treated with the LXR inhibitor GSK2033 (GSK). Red dots represent genes with a −log( P value) >1. Blue dots represent genes with expression levels below this threshold. Three hMDM donors were treated with the same plaque extract. C , Analysis of cytokine production in atherosclerotic plaque extract–conditioned hMDMs treated with the LXR inhibitor (GSK) or vehicle (DMSO) by cytometric beads array (CBA) or ELISA. Each dot represents the average response of a macrophage donor (n hMDM =6: CXCL8 [C-X-C motif ligand 8], IL [interleukin]-6, TNFα [tumor necrosis factor-α], and IL-10; n hMDM =5: IL-18BP) treated with 3 to 6 different plaques. Bars represent mean with SD. * P <0.05; Wilcoxon test was performed. D , Western blot analysis of intracellular pro-IL-1β in hMDMs from 1 donor treated with different plaque extracts, with vehicle (DMSO) or with LXR inhibitor (GSK). β-actin was used as a loading control. Each dot represents the response of the same hMDM donor treated with 5 different plaques. ** P <0.01; variables passed the Shapiro-Wilk normality test and were analyzed with a paired t test. E , Analysis of cytokine expression in atherosclerotic plaque extract–conditioned hMDMs treated with the LXR inhibitor (GSK) or vehicle (DMSO) by RT-qPCR (quantitative reverse transcription polymerase chain reaction). Each dot represents the average response of a macrophage donor (n hMDM =6) treated with 3 to 6 different plaques; bars represent mean with SD. * P <0.05, Wilcoxon test was performed. F , Analysis of cytokine expression in atherosclerotic plaque extract–conditioned hMDMs from 1 donor, conditioned with 5 different plaque extracts and treated with LXR inhibitor (SR9238) or vehicle (DMSO) by RT-qPCR; bars represent mean with SD. * P <0.05, Wilcoxon test was performed. G , Analysis of cytokine secretion in atherosclerotic plaque extract–conditioned hMDMs from 1 donor, conditioned with 5 different plaque extracts and treated with the LXR inhibitor (SR9238) or vehicle (DMSO) by ELISA. * P <0.05, Wilcoxon test was performed. P value was calculated 2 tailed for all analyses using GSK and in 1 tailed for analysis using SR9238, as SR9238 was used to confirm the effect induced by GSK. CBA indicates cytometric beads array.

Article Snippet: On day 6, hMDMs were treated for 48 hours with 1 of the following treatments: whole atherosclerotic plaque extracts (1% v/v in cell culture medium), 10 μmol/L 27-hydroxycholesterol (27OHC; Avanti Polar Lipids; Sigma; France; catalog No. 20380-11-4), or 50 μg/mL oxidized low-density lipoprotein, with or without LXR antagonist: 1 μmol/L of GSK (Sigma-Aldrich, St. Louis, MO; catalog No. 1221277-90-2) or 10 μmol/L of SR9238 (MedChemExpress, Monmouth Junction, NJ; catalog No. HY-101442).

Techniques: RNA Sequencing, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation

doi: 10.1161/ATVBAHA.125.322448

Figure Lengend Snippet: Effect of LXR (liver X receptor) inhibition on promoter accessibility of IL (interleukin)-1β, IL-6, and TNFα (tumor necrosis factor-α) genes. Graphical representation of ATAC-seq (assay for transposase-accessible chromatin using sequencing) data for IL-6, TNFα, and IL-1β genes, comparing chromatin accessibility in an human monocyte-derived macrophage (hMDM) donor treated with atherosclerotic plaque extracts (blue) vs an hMDM donor treated with atherosclerotic plaque extracts and the LXR inhibitor GSK2033 (red). The dotted area represents the gene’s promoter region. Highlighted sequences correspond to ATF (activating transcription factor)/CREB (cAMP response element-binding protein; pink), NF-κB (nuclear factor-κB; yellow), and hypoxia-response element (HRE; blue) response elements. n hMDM =1, n Plaque =1.

Article Snippet: On day 6, hMDMs were treated for 48 hours with 1 of the following treatments: whole atherosclerotic plaque extracts (1% v/v in cell culture medium), 10 μmol/L 27-hydroxycholesterol (27OHC; Avanti Polar Lipids; Sigma; France; catalog No. 20380-11-4), or 50 μg/mL oxidized low-density lipoprotein, with or without LXR antagonist: 1 μmol/L of GSK (Sigma-Aldrich, St. Louis, MO; catalog No. 1221277-90-2) or 10 μmol/L of SR9238 (MedChemExpress, Monmouth Junction, NJ; catalog No. HY-101442).

Techniques: Inhibition, Sequencing, Derivative Assay, Binding Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation

doi: 10.1161/ATVBAHA.125.322448

Figure Lengend Snippet: LXR (liver X receptor) inhibition induces the NF-κB (nuclear factor-κB) pathway. A , Fluorescent microscopy images showing atherosclerotic plaque extract–treated and vehicle-treated human monocyte-derived macrophages (hMDMs; plaque+DMSO), and atherosclerotic plaque extract–treated and LXR inhibitor–treated hMDMs (plaque+GSK2033 [GSK]). Nuclei were stained with DAPI (4′6-diamidino-2-phenylindole; blue), and NF-κB was stained with a specific antibody (green). B , Cell total corrected fluorescence of NF-κB compared between hMDMs treated with atherosclerotic plaque extracts alone (plaque+DMSO) and those treated with atherosclerotic plaque extracts and LXR inhibitor (plaque+GSK) from 1 donor treated with 5 different atheroma plaques separately. * P <0.05; variables passed the Shapiro-Wilk normality test and were analyzed with a paired t test. C , Western blot analysis of total p65 and phosphorylated p65 forms of NF-κB from hMDMs treated with vehicle (DMSO), LXR inhibitor (GSK), plaque extracts and vehicle (plaque extracts), and both LXR inhibitor and plaque extracts (plaque extracts+GSK) during 4 hours. β-Actin was used as a loading control. Graphics represent, from left to right , total p65 relative protein levels, phosphorylated p65 relative protein, and phosphorylated p65/total p65 ratio. Each dot represents the response of the same hMDM donor treated with 5 different plaques. * P <0.05, *** P <0.001; variables passed the Shapiro-Wilk normality test and were analyzed with a paired t test. D , Western blot analysis of total p65 and phosphorylated p65 forms of NF-κB from hMDMs treated with vehicle (DMSO), LXR inhibitor (GSK), plaque extracts and vehicle (plaque extracts), and both LXR inhibitor and plaque extracts (plaque extracts+GSK) during 24 hours. β-Actin was used as a loading control. Graphics represent, from left to right , total p65 relative protein levels, phosphorylated p65 relative protein, and phosphorylated p65/total p65 ratio. Each dot represents the response of the same hMDM donor treated with 5 different plaques. * P <0.05; variables passed the Shapiro-Wilk normality test and were analyzed with a paired t test. p-p65 indicates phosphorylated-p65.

Article Snippet: On day 6, hMDMs were treated for 48 hours with 1 of the following treatments: whole atherosclerotic plaque extracts (1% v/v in cell culture medium), 10 μmol/L 27-hydroxycholesterol (27OHC; Avanti Polar Lipids; Sigma; France; catalog No. 20380-11-4), or 50 μg/mL oxidized low-density lipoprotein, with or without LXR antagonist: 1 μmol/L of GSK (Sigma-Aldrich, St. Louis, MO; catalog No. 1221277-90-2) or 10 μmol/L of SR9238 (MedChemExpress, Monmouth Junction, NJ; catalog No. HY-101442).

Techniques: Inhibition, Microscopy, Derivative Assay, Staining, Fluorescence, Western Blot, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of LXR Signaling in Human Foam Cells Impairs Macrophage-to-Endothelial Cell Cross Talk and Promotes Endothelial Cell Inflammation

doi: 10.1161/ATVBAHA.125.322448

Figure Lengend Snippet: Supernatant from human monocyte-derived macrophages (hMDMs) conditioned with plaque homogenates and LXR (liver X receptor) antagonist induces a proinflammatory state in endothelial cells. A , Schematic representation of the procedure used to treat endothelial cells with conditioned macrophage supernatants. B , Left , Histograms displaying ICAM-1 (intercellular adhesion molecule-1) fluorescence levels in endothelial cells cultured with supernatants from DMSO-treated hMDMs, plaque-treated MDMs, and plaque+GSK2033 (GSK)-treated MDMs. Right , Comparison of ICAM-1 mean fluorescence intensity (MFI) in human umbilical vein endothelial cells (HUVECs) treated with supernatants from hMDMs conditioned with various plaque extracts, either alone (blue points) or in combination with an LXR inhibitor (GSK; red points). Each point represents the average effect of supernatants from 6 different hMDMs, conditioned with 3 to 6 different plaque extracts, on HUVECs. Bars represent mean with SD. * P <0.05, Wilcoxon test was performed. C , Left , Histograms displaying VCAM (vascular cell adhesion molecule) fluorescence levels in endothelial cells cultured with supernatants from DMSO-treated hMDMs, plaque-treated MDMs, and plaque+GSK-treated MDMs. Right , Comparison of VCAM mean fluorescence intensity (MFI) in HUVECs treated with supernatants from hMDMs conditioned with various plaque extracts, either alone (blue points) or in combination with an LXR inhibitor (GSK; red points). Each point represents the average effect of supernatants from 6 different hMDMs, conditioned with 3 to 6 different plaque extracts, on HUVECs. Bars represent mean with SD. * P <0.05, Wilcoxon test was performed. D and E , Comparative analysis of relative mRNA levels of ICAM-1 ( D ) or CCL2 (C-C motif ligand 2; E ) by RT-qPCR (quantitative reverse transcription polymerase chain reaction) in HUVECs treated with supernatants from hMDMs conditioned with various plaque extracts, either alone (blue points) or in combination with an LXR inhibitor (GSK; red points). Each point represents the average effect of supernatants from 7 different hMDMs, conditioned with 3 to 6 different plaque extracts, on HUVECs. Bars represent mean with SD. * P <0.05, Wilcoxon test was performed. F and G , Relative mRNA levels of targeted gene (ICAM-1 [ F ] and CCL2 [ G ]) in HUVECs treated with DMSO-treated hMDM supernatant, atherosclerotic plaque extract–treated hMDM supernatant, atherosclerotic plaque extract–treated and LXR inhibitor– treated hMDM supernatant, and atherosclerotic plaque extract–treated and LXR inhibitor–treated hMDM supernatant with tocilizumab (40 µmol/L). Each dot represents the response of supernatants from 12 different hMDMs, conditioned with a mix of 6 different plaques, on HUVECs. Bars represent mean with SD. * P <0.05, ** P <0.01, *** P <0.001; Friedman test was performed with uncorrected Dunn post hoc test for multivariate analyses. M-CSF indicates macrophage colony stimulating factor; and TCZ, tocilizumab.

Article Snippet: On day 6, hMDMs were treated for 48 hours with 1 of the following treatments: whole atherosclerotic plaque extracts (1% v/v in cell culture medium), 10 μmol/L 27-hydroxycholesterol (27OHC; Avanti Polar Lipids; Sigma; France; catalog No. 20380-11-4), or 50 μg/mL oxidized low-density lipoprotein, with or without LXR antagonist: 1 μmol/L of GSK (Sigma-Aldrich, St. Louis, MO; catalog No. 1221277-90-2) or 10 μmol/L of SR9238 (MedChemExpress, Monmouth Junction, NJ; catalog No. HY-101442).

Techniques: Derivative Assay, Fluorescence, Cell Culture, Comparison, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Nature Communications

Article Title: An ESCRT-dependent step in fatty acid transfer from lipid droplets to mitochondria through VPS13D−TSG101 interactions

doi: 10.1038/s41467-021-21525-5

Figure Lengend Snippet: a Purified VPS13D-LTD was incubated with fluorescent lipids and examined by native PAGE. Phospholipids and FAs, visualized by their fluorescence, comigrated with protein, visualized by coomassie blue staining. PA: phosphatidic acid; PS: phosphatidylserine; PE: phosphatidylethanolamine; PC: phosphatidylcholine; Cer: ceramide; C12: NBD-C12; C16: BODIPY-C16. b Fold change of lipid fluorescence to protein. Three biological replicates were performed with similar results. Ordinary one-way ANOVA (Tukey’s multiple comparisons test). **** p < 0.0001. Mean ± SD. c Estimation of the stoichiometry of VPS13D-LTD binding to BODIPY-C16. d Fold change of lipid fluorescence to protein in c . Three biological replicates were performed with similar results. Mean ± SD. e Competition assays showing that unlabeled, non-BSA-conjugated palmitic acid, but not OA, sufficiently outcompeted BODIPY-C16 for binding to VPS13D-LTD. f Fold change of lipid fluorescence to protein in e . Three biological replicates were performed with similar results. Mean ± SD. g Quantification of Red C12 fluorescence in mitochondria upon 5 h of starvation. For t = 0: cells treated with scrambled ( n = 16), VPS13D ( n = 18), VPS13D siRNA/siRNA resistant VPS13D^sfGFP ( n = 11), VPS13D siRNA/siRNA resistant GFP-LTD-WT ( n = 11), VPS13D siRNA/siRNA resistant GFP-LTD-mut ( n = 12) were analyzed. For t = 5 h: cells treated with scrambled ( n = 21), VPS13D ( n = 18), VPS13D siRNA/siRNA resistant VPS13D^sfGFP ( n = 10), VPS13D siRNA/siRNA resistant GFP-LTD-WT ( n = 14), VPS13D siRNA/siRNA resistant GFP-LTD-mut ( n = 11) were analyzed. For t = 24 h: scrambled ( n = 11), VPS13D ( n = 13), VPS13D siRNA/siRNA resistant VPS13D^sfGFP ( n = 15), VPS13D siRNA/siRNA resistant GFP-LTD-WT ( n = 18), VPS13D siRNA/siRNA resistant GFP-LTD-mut ( n = 15) were analyzed. A.U., arbitrary unit. Three independent assays were performed with similar results. Ordinary one-way ANOVA (Tukey’s multiple comparisons test) within five groups in category of t = 5 h or t = 24 h. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: p > 0.05. Mean ± SD. h – l Red C12 p ulse-chase assay ( t = 5 h) of HEK293 cells treated with scrambled ( h ), VPS13D siRNAs ( i ), VPS13D siRNA/siRNA-resistant VPS13D^sfGFP ( j ), VPS13D siRNA/siRNA-resistant GFP-VPS13D-LTD-WT ( k ) or VPS13D siRNA/siRNA-resistant GFP-VPS13D-LTD-mut ( l ). Scale bar, 10 μm in whole cell image and 2 μm in insets in h – l .

Article Snippet: Lipids were purchased from Avanti Polar Lipids: NBD-PC (810133), NBD-PE (810144), NBD-PS (810198), NBD-PA (810138), NBD-ceramide (810211).

Techniques: Purification, Incubation, Clear Native PAGE, Fluorescence, Staining, Binding Assay