Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: Incubation with MβCD diminished cholesterol content in fibroblasts.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Incubation

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: Representative images of actin cytoskeleton from fibroblasts untreated (A–C) or treated with either 5 (D–F) or 10 (G–I) mM of MβCD for 45 minutes at 37°C, fixed and labeled with Phalloidin-FITC and DAPI (cell nuclei). Three independent experiments were performed in triplicates and at least 50 different cells were observed per group. Each selected image represents the most predominant actin cytoskeleton cell morphology of that experimental condition. Scale bars are all 10 µm.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Labeling

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: Fibroblasts, treated with MβCD 10 mM, for 20 or 40 minutes, were submitted to Rho-GTP pull down assay. In (A), left side, we have the western-blotting result of the pull-down eluates showing that after 20 or 40 minutes of cholesterol removal Rho is activated in fibroblasts. In (A), right side, we have cell lysates that were not submitted to the pull-down assay showing the total amount of Rho present in each experimental group. Control untreated fibroblasts and cell extract incubated with GDP were used as controls of the Rho activation and of the pull-down technique, respectively. (B) Graph showing the densitometric analysis of activated Rho seen in the autoradiogram of the upper panel (A) normalized by the corresponding band at the cell lysates. The data are mean ± standard error of two independent experiments. Data are expressed in arbitrary units (folds in relation to control).

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Pull Down Assay, Western Blot, Control, Incubation, Activation Assay

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: Fibroblasts were submitted to the following treatments: 95 nM of Latrunculin-A (Lat A) for one hour followed by washing with HBSS+ and incubation with fresh DMEM, without serum, for 45 minutes (A–C); previous incubation with Lat-A followed by washing with HBSS+ and incubation with 5 (D–F) or 10 (G–I) mM of MβCD diluted in fresh DMEM, without serum, for 45 minutes. The cells were fixed simultaneously and processed for F-actin cytoskeleton labeling using Phalloidin-FITC. Cell nuclei were stained with DAPI. Three independent experiments were performed in triplicates and at least 50 different cells were observed per group. Each selected image represents the most predominant actin cytoskeleton cell morphology of that experimental condition. Scale bars are all 10 µm.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Incubation, Labeling, Staining

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: (A) Typical image of a tether extracted from a fibroblast, indicated with a white arrowhead. Scale bar is 5 µm. (B) Typical force versus displacement plot showing the plateau corresponding to F 0 . (C) Plot representing the mean values of the force F 0 for the control, 5 and 10 mM of MβCD. (D) Representative image of a tether obtained by scanning electron microscopy. Scale bar is 1 µm. (E) Plot representing the mean values of the radii obtained for control 5 and 10 mM of MβCD. Three independent experiments were performed and at least 20 different cells were analyzed per each group. The plotted results represent the mean ± standard error of all the measurements. ns means no significant difference and ** means p<0.01 in student t-test.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Control, Electron Microscopy

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: Cholesterol depletion increased surface tension and bending modulus.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques:

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: (A) Representative DM image showing a control untreated fibroblast. Scale bar: 20 µM. (B) The region of the inset in (A) shows control case. (C–G) Same region of the inset in (B) at different stages of cyclodextrin treatment. Temporal correlation functions for each corresponding time interval are also displayed under each representative images. Scale bar: 10 µM.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Control

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: Images of untreated cells were collected during 10 minutes. Following it, cyclodextrin was added and images continued to be collected for the subsequent 45 minutes. Images were analyzed and the relaxation time τ (A and B) and the amplitude of curvature A (C and D) of fibroblasts treated either with 5 (A and C) or 10 mM (B and D) of MβCD was obtained according to the equation above graphs. Five independent experiments were performed and at least three regions of each one of the five cells were analyzed. The plotted data represent a mean ± standard error of these five independent experiments. Different letters indicate statistically significant differences (p<0,05 using One-way ANOVA and Newman-Keuls post-test).

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques:

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: (A) Lysosomal exocytosis assay of fibroblasts exposed to 10 mM of MβCD for 10, 20 or 40 minutes. Ionomycin was used as a positive control for lysosomal exocytosis. Asterisks (*) indicate statistically significant differences between control and treated groups (p<0.05 using student t-test). (B) Knocking down of syt-VII in NRK cells was confirmed by RT-PCR, using sytVII or actin specific primers. Left: Control non-transfected NRK (control), NRK transfected with medium GC siRNA – negative control oligo (NC oligo) and NRK transfected with syt-VII siRNA oligo (siRNA sytVII). Right: quantification of the intensity of the gel bands (C) Lysosomal exocytosis assay of non-transfected NRK (control cells), NRK transfected with medium GC siRNA – negative control oligo (NC oligo) and NRK transfected with syt-VII siRNA oligo (siRNA sytVII). Asterisk (*), hashtag (#) and diamond (υ) indicate statistically significant differences between MβCD treated groups and their respective controls (p<0.05 using student t-test). (D) Lysosomal exocytosis assay of fibroblasts pre-treated or not with Lat-A 95 nM, for one hour, followed or not by incubation with MβCD 10 mM for 10, 20 or 40 minutes. Asterisks (*) and hashtags (#) indicate statistically significant differences between control and MβCD treated groups (*) or Lat-A and Lat-A+MβCD treatment (#) (p<0.05 using student t-test). (A, C and D) The results are expressed as the ratio between β-hex activity in the cell supernatant and total β-hex activity and are shown as mean of triplicates ± standard error. Three independent experiments were performed in triplicates. Pooled data are shown as mean of all results ± standard error.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Positive Control, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Incubation, Activity Assay

Journal: PLoS ONE

Article Title: Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

doi: 10.1371/journal.pone.0082988

Figure Lengend Snippet: (A–C) Representative images of control, MβCD 10 mM treated and Lat-A+MβCD 10 mM treated cells, fixed and immunolabeled for DAPI and LAMP-1. PC: phase contrast. Scale bar: 10 µm. (D–E) Quantitative analysis of lysosomal distribution, relative to cell nuclei, in MβCD 10 mM (D) and Lat-A 95 nM+MβCD 10 mM (E) in comparison to control non-treated cells. The mean distance between a lysosome and its respective cell center is represented by letter D and the mean lysosome distance relative to the mean nucleus’ radius R was defined as the ratio D/R . The histogram for the control (non-treated) lysosomal distribution is represented with a red solid thick line, MβCD 10 mM is represented with a blue dashed line and Lat-A+MβCD combined treatment is represented with a green dashed line. Overlaid Gaussian fits to each distribution were added in order to highlight the main statistical trend of each diagram. The x-axis of each graph represents the ratio D/R whereas the y-axis represents the percentage of analyzed nuclei from each treatment normalized by total cell number. (F) Cumulative frequency of the histograms. Plus sign blue line represents MβCD 10 mM treated cells, dashed green line represent Lat-A+MβCD 10 mM treated cells and red continuous line represent control untreated cells. The Kolmogorov- Smirnov (KS) statistical test was performed to compare the cumulative frequency distributions. Statistically significant differences were obtained for MβCD 10 mM 0.25% (p = 0.0025), Lat-A+MβCD 10 mM 0.000091% (p = 0.00000091) treated cells in relation to control, and for MβCD 10 mM in relation to Lat-A+MβCD 10 mM 4,3% (p = 0.043). (G) Cell area comparison between control and treated groups. No statistical differences were detected among them (p>0.05 student T-test). Three independent experiments were performed in triplicates and at least 20 cells from each condition were analyzed. Data are shown as mean of at least 20 cells from a representative experiment ± standard error.

Article Snippet: For plasma membrane cholesterol sequestration, cells were first washed three times with Hank’s Balanced Salt Solution supplemented with 1.8 mM of CaCl 2 .2H 2 O (HBSS +), and then incubated with 5 or 10 mM methyl-beta cyclodextrin (MβCD) solutions diluted in DMEM high glucose (Invitrogen, Carlsbad, CA, USA), in the absence of serum, for 45 minutes at 37°C and 5% CO 2 .

Techniques: Control, Immunolabeling, Comparison

Journal: iScience

Article Title: Elaidic acid drives cellular senescence and inflammation via lipid raft-mediated IL-1R signaling

doi: 10.1016/j.isci.2025.113305

Figure Lengend Snippet: EA preferentially accumulates in the lipid rafts, thereby potentiating the activation of the IL-1R signaling (A) U2OS cells were pretreated with or without 200 μM EA for 12 h, and then overexpressed with Flag-MyD88 for 24 h, and assayed for NF-κB luciferase activity. Data are shown as mean ± SD ( n = 3). (B and C) U2OS cells were pretreated with or without 200 μM EA for 12 h, and then stimulated with 10 ng/mL IL-1α for 12 h, subjected to qRT-PCR analysis. Relative mRNA levels of IL-6 (b) and IL-8 (c) are shown as mean ± SD ( n = 3). (D) U2OS cells were pretreated with or without 200 μM EA for 12 h, and then stimulated with 10 ng/mL IL-1α for the indicated time periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. (E) U2OS cells were pretreated with or without 200 μM EA for 12 h, treated with MβCD (5 mM) for 0.5 h, and then stimulated with 10 ng/mL IL-1α for the indicated time periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. (F) Immunoblot analysis of DRM fraction. The four fractions from density gradient ultracentrifugation were analyzed by immunoblotting using anti-Flotillin-1 (DRM fraction corresponding to lipid rafts) and Calnexin (non-DRM fraction corresponding to endoplasmic reticulum, and so forth) antibody. (G and H) Ratios of tetra-deuterated EA and OA incorporated into phosphatidylcholine (PC) 16:0-18:1 and 18:0-18:1 in Flotillin-1- (g) and Calnexin- (h) positive fractions. Data are shown as mean ± SD ( n = 3). (I and J) U2OS cells were treated with 200 μM EA for 12 h, and subjected to immunoblotting with the indicated antibodies (i). The band intensity of IL-R was normalized with that of β-actin, and shown as mean ± SD ( n = 3) (j). (K and L) U2OS cells were treated with 200 μM EA or LEA for 12 h, followed by density gradient ultracentrifugation. The samples were separated into four fractions and analyzed by immunoblotting with the indicated antibodies (L). The band intensity of IL-R in Fr. 1 was quantified and expressed as a percentage of the total band intensity across all fractions. Data are represented as mean ± SD ( n = 4). (M) U2OS cells were pretreated with 200 μM EA for 12 h, treated with ACSL inhibitor Triacsin C (0.5 μM), and then stimulated with 10 μM CDDP for 48 h, subjected to qRT-PCR analysis. Relative mRNA levels of IL-8 are shown as mean ± SD ( n = 3).

Article Snippet: Methyl-beta-cyclodextrin (MβCD) , Selleckchem , Cat#S6827.

Techniques: Activation Assay, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Cell stem cell

Article Title: Phospholipid remodeling and cholesterol availability regulate intestinal stemness and tumorigenesis

doi: 10.1016/j.stem.2017.12.017

Figure Lengend Snippet:

Article Snippet: Methyl-β-cyclodextrin , Cyclodextrin Technologies Inc , TRMB-P.

Techniques: Plasmid Preparation, Recombinant, Amplex Red Cholesterol Assay, Microarray, Generated, Transgenic Assay, Software

Journal: The Journal of Cell Biology

Article Title: MT1-MMP recruits the ER-Golgi SNARE Bet1 for efficient MT1-MMP transport to the plasma membrane

doi: 10.1083/jcb.201808149

Figure Lengend Snippet: MT1-MMP is located in proximity to Bet1 in a raft-like, cholesterol-rich membrane domain. (A) Depletion of STX4 and Vti1b reduces the PLA signals between MT1-MMP and Bet1-GFP. MDA-MB-231 cells stably expressing Bet1-GFP were transfected with STX4 and Vti1b siRNAs, and the PLA was performed as in . Arrows indicate accumulated PLA signals in endosomes, whereas asterisks indicate nonspecific staining in nuclei. (B) Quantitation of PLA signals between MT1-MMP and Bet1-GFP as in A. (C) Cholesterol depletion weakens the interplay between MT1-MMP and Bet1. MDA-MB-231 cells stably expressing Bet1-GFP were spread on a fibronectin-coated coverslip for 7 h and then treated with 0.1% DMSO (vehicle), 5 mM methyl-β-cyclodextrin (MβCD) or 50 µg/ml nystatin for 30 min. After that, the cells were fixed and then subjected to PLA for MT1-MMP and Bet1-GFP. (D) Quantitation of PLA signals between MT1-MMP and Bet1-GFP as in C. (E) Depletion of Cav1 abolishes the proximity between MT1-MMP and Bet1-GFP. The experiments were performed as in A except for the use of Cav1 siRNA. (F) Quantitation of PLA signals between MT1-MMP and Bet1-GFP as in E. Scale bar: 10 μm. *, P < 0.05; **, P < 0.01; vs. mock in B and F; vs. vehicle in D.

Article Snippet: Briefly, cells were spread on fibronectin-coated coverslips and left for 7 h, and then treated with 5 μM methyl-β-cyclodextrin (Wako Pure Chemicals) or 50 μg/ml nystatin (Sigma-Aldrich) for 30 min at 37°C.

Techniques: Stable Transfection, Expressing, Transfection, Staining, Quantitation Assay