Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 1 Veklury® (remdesivir) formulations induce alterations in membrane structure and disrupt lipid rafts similarly to cyclodextrins (CDs) in a cholesterol-dependent manner independently of remdesivir. (a) Control HEK/ACE2 + TMPRSS2 cells and those treated with 1-, 5- or 10-mM MβCD, HPβCD, SBECD, Veklury® P (powder) or Veklury® S (solution), 100-μM remdesivir (REM) or 200-μM cholesterol (CHOL) were labelled with cholera toxin subunit B (CTX-B), a lipid raft marker, and N-[3-(40-dihexylamino-3-hydroxy-flavonyl-6-oxy)-propyl]N,N-dimethyl-N- (3-sulfopropyl)-ammonium inner salt (F66), an environment-sensitive fluorescent indicator. Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverslip show F66 intensities of the normal (N*) and tautomeric (T*) excited states and the T*/N* emission ratios calculated pixel by pixel. Cell membrane masks selected manually in CTX-B images were segmented using the maxentropy algorithm to ‘raft’ (CTX-B high) and ‘non-raft’ (CTX-B low) regions corresponding to high and low intensities of the raft markers, respectively, and average T*/N* intensity ratios were determined separately for the two masks (symbols in [c]). (b) Pixelwise distributions of the T*/N* F66 emission ratio in CTX-B high ‘raft’ and CTX-B low ‘non-raft’ regions of control cells are displayed. As an alternative definition for raft regions, a threshold value of the T*/N* ratio was determined (red dashed line) and membrane pixels were considered as ‘raft’ and ‘non-raft’ regions when being above and below the threshold, respectively (evaluation shown by the bars in [c]). The threshold value was chosen to result in a segmentation pattern similar to that obtained in (a). (c) The average F66 T*/N* ratios calculated for CTX-B high ‘raft’ and CTX-B low ‘non-raft’ pixels (circles and squares, respectively; numerical values to be read on the left vertical axis) as well as the relative fraction of ‘raft’ pixels (bars, numerical values to be read on the right vertical axis) above the F66 emission ratio threshold (shown by the red line in [b]) were determined in individual cells (n = 20) and the mean ± SD values are plotted. Asterisks indicate significant differences compared to the control samples (*P < 0.05; one-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S1.

Article Snippet: HEK/ACE2 + TMPRSS2 cells grown on eight-well chambered coverslip (ibidi, Gräfelfing, Germany; 80826) were treated for 1 h at room temperature with MβCD, HPβCD, SBECD, Veklury® P or Veklury® S at CD concentrations of 1, 5 and 10 mM, with cholesterol complexed with MβCD (CHOL) at 200 μM or pure remdesivir (GS-5374; REM) at 100 μM.

Techniques: Membrane, Control, Marker

Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 2 Methyl-β cyclodextrin (MβCD)-induced reductions in ACE2 binding of Wuhan-Hu-1 strain of SARS-CoV-2 (WT) and Delta SARS- CoV-2 spike receptor-binding domains (RBDs) negatively correlate with the applied RBD concentrations. HEK/ACE2 + TMPRSS2 cells were treated with different concentrations of MβCD ranging between 0.25 and 10 mM. Fluorescence intensities of cells were measured using flow cytometry for 5 min continuously starting immediately after the addition of GFP-conjugated WT (a) or Delta (b) SARS-CoV-2 spike RBDs applied at various concentrations between 0.1 and 5 μgml1, or 0.05 and 2.5 μgml1, respectively. Moving averages of time-correlated fluorescence intensities of at least 50,000 cells of normal morphology were determined with a window size of 10 s and subsequently normalized to the average of intensities obtained in the last time window of untreated control samples. Mean ± SD values were calculated from data of six independent samples per treatment condition and plotted as a function of time (starting 20 s after RBD addition). The extents of inhibition of WT (a) and Delta (b) RBD binding at the end of the measurement time in response to various MβCD concentrations were calculated and plotted as mean ± SD as a function of the applied RBD concentration in the right panels. Asterisks indicate significant differences of samples treated with the lowest versus highest RBD concentrations at each applied CD concentration (*P < 0.05; two-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S2.

Article Snippet: HEK/ACE2 + TMPRSS2 cells grown on eight-well chambered coverslip (ibidi, Gräfelfing, Germany; 80826) were treated for 1 h at room temperature with MβCD, HPβCD, SBECD, Veklury® P or Veklury® S at CD concentrations of 1, 5 and 10 mM, with cholesterol complexed with MβCD (CHOL) at 200 μM or pure remdesivir (GS-5374; REM) at 100 μM.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Control, Inhibition, Concentration Assay

Journal: British journal of pharmacology

Article Title: Veklury® (remdesivir) formulations inhibit initial membrane-coupled events of SARS-CoV-2 infection due to their sulfobutylether-β-cyclodextrin content.

doi: 10.1111/bph.16063

Figure Lengend Snippet: FIGURE 3 Veklury® (remdesivir) formulations and cyclodextrin (CDs) reduce the cellular binding of Wuhan-Hu-1 strain of SARS-CoV-2 (WT) and Delta SARS-CoV-2 spike receptor-binding domain (RBDs) in a cholesterol-dependent manner. HEK/ACE2 + TMPRSS2 (a) or Calu-3 (b) cells were treated with a dilution series of MβCD, HPβCD, SBECD, Veklury® P (powder), Veklury® S (solution) or a 1:1 (w/w) mixture of Veklury® P and SBECD at CD concentrations ranging between 0.078 and 20 mM, remdesivir (REM) between 75 and 1200 μM or cholesterol (CHOL) between 12.5 and 800 μM. HEK/ACE2 + TMPRSS2 cells were incubated in the presence of 0.2-μgml1 WT or 0.1-μgml1 Delta SARS-CoV-2 spike RBD–GFP, whereas Calu-3 cells were labelled with 1-μgml1 WT or 0.5-μgml1 Delta RBD–GFP for 4 min. The emitted fluorescence intensities of individual cells were subsequently measured using flow cytometry and the mean intensity was calculated from data of at least 10,000 cells of normal morphology per sample. The average values of six independent measurements (±SD) were calculated, normalized to the mean value determined in untreated control samples and plotted as a function of the applied concentrations of CD (in the left panels) and CHOL or REM (in the right panels). Asterisks indicate significant differences of samples treated with the highest applied concentrations compared to the control samples (*P < 0.05; two-way ANOVA followed by Tukey's HSD test). See details of statistical analysis in Table S3.

Article Snippet: HEK/ACE2 + TMPRSS2 cells grown on eight-well chambered coverslip (ibidi, Gräfelfing, Germany; 80826) were treated for 1 h at room temperature with MβCD, HPβCD, SBECD, Veklury® P or Veklury® S at CD concentrations of 1, 5 and 10 mM, with cholesterol complexed with MβCD (CHOL) at 200 μM or pure remdesivir (GS-5374; REM) at 100 μM.

Techniques: Binding Assay, Incubation, Fluorescence, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) Schematic of DIBMALP containing an EphA2-GFP monomer. (B) Step distribution of control DIBMALPs (black) or those formed from cells treated with EA1 (pink), MβCD (blue) or both (magenta). (C) Oligomeric distribution calculated from data in panel B. (D) Schematic representing DDM micelles containing EphA2-GFP. (E) Step distribution of DDM-solubilized EphA2-GFP in the same conditions as in DIBMALPs. (F) Oligomeric distribution of DDM-solubilized EphA2-GFP photobleaching data. p -values are from two-way ANOVA followed by Tukey multiple comparison test.*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Methyl-β-cyclodextrin (MβCD) (Acros Organics) was used to remove cholesterol from cultured cells.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Cholesterol inhibits assembly and activation of the EphA2 receptor

doi: 10.1101/2024.06.10.598255

Figure Lengend Snippet: (A) cAMP quantification in HEK293T cells transduced with Red Up cADDis cAMP biosensor ollowing treatment with MβCD. (B) Western blot analysis and quantification of PKA T197 phosphorylation in A375 cells following treatment with MβCD. (C-D) Western blot analysis and quantification of EphA2 S897 phosphorylation in A375 cells following treatment with forskolin and soproterenol, respectively. Data shown in panel B are normalized to the respective total PKA signal in Figure S15A. Data shown in panels C-D are normalized to the respective total EphA2 signal in Figure S15B-C. Quantitative comparisons between treatments were made with respect to normalized control conditions. Bar graphs show mean ± S.D., p -values in panels A-D are from an unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

Article Snippet: Methyl-β-cyclodextrin (MβCD) (Acros Organics) was used to remove cholesterol from cultured cells.

Techniques: Transduction, Western Blot, Phospho-proteomics, Control

Journal: Cancer research

Article Title: Characterizing macrophage diversity in metastasis-bearing lungs reveals a lipid-associated macrophage subset

doi: 10.1158/0008-5472.CAN-21-0101

Figure Lengend Snippet: (A-E) Feature and violin plots of key genes demonstrate that macrophages isolated from metastasis-bearing lungs separate into unique clusters defined by gene signatures related to (A) antigen presentation, (B) cell cycle and proliferation, (C) interferon responsiveness, (D) immunosuppressive, pro-tumorigenic immune responses and matrix remodeling, and (E) cholesterol and lipid metabolism.

Article Snippet: Following generation and overnight attachment, cells were starved for one hour in media containing 1% FBS (Atlanta Biologicals) and loaded with 40μg/mL soluble cholesterol (1:6 with methyl-β-cyclodextran in 0.2% BSA in RPMI) or 100μg/mL oxLDL (Thermo Fisher), or left unloaded as controls.

Techniques: Expressing, Isolation, Immunopeptidomics

Journal: Cancer research

Article Title: Characterizing macrophage diversity in metastasis-bearing lungs reveals a lipid-associated macrophage subset

doi: 10.1158/0008-5472.CAN-21-0101

Figure Lengend Snippet: (A) Waterfall plots demonstrate that, compared to other macrophage clusters, lipid-associated macrophages in metastasis-bearing lungs (cluster 6) demonstrate highly similar gene signatures to defined bulk and scRNA-seq datasets of foamy macrophages isolated from atherosclerotic plaques. scRNAseq_foamy_UP_nonfoamy_DOWN (NES=1.85, p adj=0.002), Bulk_RNAseq_foamy_UP_nonfoamy_DOWN, (NES=1.91, p adj=0.002), Bulk_RNAseq_foamy_DOWN_nonfoamy_UP, (NES=−2.06, p adj=0.014), scRNAseq_foamy_DOWN_nonfoamy_UP, (NES = −1.80, p adj=0.014). (B) Heatmap of relative expression among metastasis-associated macrophage subsets of core enrichment genes defining foamy macrophages based on a published scRNA-seq dataset ( 17 ). (C-E) GSEA of cluster 6 versus other macrophage clusters (C0–3, 5, 8) identified by scRNA-seq demonstrates positive enrichment for genes implicated in pathways related to lipid metabolism. Waterfall plots showing relative enrichment of genes involved in (C) lysosome function (NES=1.79, p adj=0.12, FDR=0.11) (D) cholesterol efflux (NES=1.62, p adj=0.17, FDR=0.15) and (E) lipid catabolism (NES=1.50, p adj=0.15, FDR=0.13) in cluster 6 compared to other macrophage clusters. NES = normalized enrichment score.

Article Snippet: Following generation and overnight attachment, cells were starved for one hour in media containing 1% FBS (Atlanta Biologicals) and loaded with 40μg/mL soluble cholesterol (1:6 with methyl-β-cyclodextran in 0.2% BSA in RPMI) or 100μg/mL oxLDL (Thermo Fisher), or left unloaded as controls.

Techniques: Gene Expression, Isolation, RNA sequencing, Expressing

Journal: Cancer research

Article Title: Characterizing macrophage diversity in metastasis-bearing lungs reveals a lipid-associated macrophage subset

doi: 10.1158/0008-5472.CAN-21-0101

Figure Lengend Snippet: (A) Intranasal (i.n.) administration of clodronate liposomes results in a decrease in the relative frequency of alveolar macrophages compared to interstitial macrophages (n=5 mice/group). (B) Alveolar macrophage depletion results in a reduction in the number of SSC hi BODIPY hi cells per gram of tissue among total macrophages and alveolar macrophages (n=5 mice/group). (C) The frequency of alveolar macrophages exhibiting positive BODIPY 493/503 staining increases following liposomal clodronate depletion compared to controls (n=5 mice/group). (D) BMDMs loaded with soluble cholesterol or oxLDL in culture exhibit decreased phagocytosis of Zymosan particles compared to non-lipid-loaded controls. Data are shown as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 as determined by ANOVA followed by Sidak’s or Dunnett’s multiple comparisons test.

Article Snippet: Following generation and overnight attachment, cells were starved for one hour in media containing 1% FBS (Atlanta Biologicals) and loaded with 40μg/mL soluble cholesterol (1:6 with methyl-β-cyclodextran in 0.2% BSA in RPMI) or 100μg/mL oxLDL (Thermo Fisher), or left unloaded as controls.

Techniques: Liposomes, Staining