Lipid accumulation controls the balance between surface connection and scission of caveolae
- Department of Integrative Medical Biology, Umeå University, Sweden
- Swedish Metabolomics Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Sweden
- Swedish Metabolomics Centre, Department of Molecular Biology, Umeå University, Sweden
- Department of Chemistry, Umeå University, Sweden
Figures
Figure 1 with 4 supplements
Rapid insertion of Bodipy-labeled lipids into the PM of living cells using fusogenic liposomes.
(A) Fusogenic liposomes are used to insert Bodipy-labeled lipids into the PM. Their rapid distribution is followed in real time using TIRF microscopy. (B) Image sequence of Bodipy-LacCer …
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Figure 1—source data 1
Excel file containing source data pertaining to Figure 1B–F.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Caveolae dynamics at the cell surface.
Scheme illustrating caveolae dynamics at PM. Caveolae formation and coat assembly are primarily driven by the integral membrane protein Cav1 and cavin proteins. EHD2 controls surface association of …
Figure 1—figure supplement 2
Liposome characterization and vesicle fusion with the PM.
(A) Chemical structures of lipids used in this study. (B) Hydrodynamic diameter as z-average of DOPE:DOTAP:Bodipy-lipid liposomes. n = 3, three independent experiments, mean ± SD. (C) Total Bodipy …
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Figure 1—figure supplement 2—source data 1
Excel file containing source data pertaining to Figure 1—figure supplement 2A–C and E.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Quantification of Bodipy-lipid incorporation using mass spectrometry.
(A, B) Visualization of chromatography of Bodipy-SM C12. Samples visualized: Bodipy-LacCer treated samples, Bodipy-SM C12 treated samples, DOPE control samples and a negative control of Bodipy-SM C12…
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Figure 1—figure supplement 3—source data 1
Excel file containing source data pertaining to Figure 1—figure supplement 3A and B.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig1-figsupp3-data1-v2.xlsx
Figure 1—figure supplement 4
Characterization of Cav1-mCh HeLa cells.
(A) Representative immunoblots showing protein expression of EHD2, Cav1-mCh, and Cav1 after induction of Cav1-mCh HeLa cells with different concentrations of Dox. Clathrin HC served as loading …
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Figure 1—figure supplement 4—source data 1
Excel file containing source data pertaining to Figure 1—figure supplement 4C.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig1-figsupp4-data1-v2.xlsx
Figure 2 with 7 supplements
GSLs and Chol decrease the surface stability of caveolae.
(A) Scheme showing different dynamic behaviors of caveolae. (B, B’) Distribution of track mean speed among subpopulations of track duration of Cav1-mCh structures (B) and after EHD2 depletion (B’). …
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Figure 2—source data 1
Excel file containing source data pertaining to Figure 2B and D–G.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Correlation between track duration and track mean speed after different treatments.
(A, B) Correlation between track duration and track mean speed. TIRF live cell movies of Cav1-mCh structures (A) and cells lacking EHD2 (B) were analyzed. Identical symbols represent tracks from the …
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Figure 2—figure supplement 1—source data 1
Excel file containing source data pertaining to Figure 2—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Effect of Bodipy-lipid addition on Cav1-mCh tracks and quantification of Cav1-mCh positive structures.
(A) Representative images from TIRF live cell movies of Dox-induced Cav1-mCh HeLa cells after incubation with different fusogenic liposomes containing Bodipy-lipids (final total lipid concentration …
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Figure 2—figure supplement 2—source data 1
Excel file containing source data pertaining to Figure 2—figure supplement 2B–D.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
Caveolae tracking with Imaris software.
To track caveolae dynamics, 5 min TIRF movies of induced Cav1-mCh HeLa cells were recorded with an acquisition time of 3 s. One frame from the movie is depicted (Cav1-mCh). Spot analysis of Cav1-mCh …
Figure 2—video 1
Cell surface dynamics of Cav1-mCh.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells. The image in Fig. 2C (Cav1-mCh) is taken from this movie. Movie in real time spans 5 min and was recorded at 3 s intervals. …
Figure 2—video 2
Cell surface dynamics of Cav1-mCh after treatment with Bodipy-LacCer.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells after 15 min incubation with liposomes containing Bodipy-LacCer. The image in Fig. 2C is taken from this movie. Movie in real …
Figure 2—video 3
Cell surface dynamics of Cav1-mCh after treatment with Bodipy-Chol.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells after 15 min incubation with liposomes containing Bodipy-Chol. The image in Fig. 2C is taken from this movie. Movie in real …
Figure 2—video 4
Cell surface dynamics of Cav1-mCh after treatment with Bodipy-SM C12.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells after 15 min incubation with liposomes containing Bodipy-SM C12. The image in Fig. 2C is taken from this movie. Movie in real …
Figure 3 with 4 supplements
Chol and GSLs induce surface release of caveolae via an EHD2-dependent mechanism.
(A) Representative images of maximum projected confocal z-stacks of Cav1-mCh HeLa cells. Untreated cells or cells treated with LacCer-Bodipy liposomes for 1 h, fixed and immunostained for endogenous …
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Figure 3—source data 1
Excel file containing source data pertaining to Figure 3A–C,E,F and H.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Cavin1 localization to Cav1-mCh before and after liposome treatment.
(A) Representative TIRF micrographs of cavin1-GFP transfected Dox-induced Cav1-mCh HeLa cells. The same cells were imaged before and after incubation with fusogenic liposomes as depicted (final …
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Figure 3—figure supplement 1—source data 1
Excel file containing source data pertaining to Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
EHD2 depletion in Cav1-mCh HeLa cells.
(A) Representative immunoblots of Cav1-mCh HeLa cells treated with Ctrl siRNA or siRNA against EHD2. Clathrin HC served as loading control. (B) Effect of lipids on track duration of Cav1-mCh …
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Figure 3—figure supplement 2—source data 1
Excel file containing source data pertaining to Figure 3—figure supplement 2B and C.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
Stabilization of caveolae to the PM by EHD2-I157Q cannot be reversed by addition of Bodipy-labeled LacCer or Chol.
(A) Representative time-lapse images of Cav1-mCh positive for EHD2-BFP (yellow arrows) or lacking EHD2-BFP (white arrows) in double Flp-In EHD2-BFP Cav1-mCh HeLa cells. Dotted box shows higher …
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Figure 3—figure supplement 3—source data 1
Excel file containing source data pertaining to Figure 3—figure supplement 3B,D and E.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig3-figsupp3-data1-v2.xlsx
Figure 3—figure supplement 4
Microinjection of EHD2-647 into Cav1-mCh HeLa cells.
(A) Representative live cell TIRF images of Cav1-mCh HeLa cells untreated or treated with Bodipy-LacCer and with or without microinjection of EHD2-647. (B) Quantification of the colocalization of …
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Figure 3—figure supplement 4—source data 1
Excel file containing source data pertaining to Figure 3—figure supplement 4B.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig3-figsupp4-data1-v2.xlsx
Figure 4 with 7 supplements
LacCer, Chol, and SM accumulate in caveolae and Chol is sequestered within these domains.
(A–D) Cav1-mCh HeLa cells transiently expressing EHD2-BFP were incubated with Bodipy-lipids. White lines indicate location of kymograph. TIRF movies were recorded at 3 s intervals for 5 min. Scale …
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Figure 4—source data 1
Excel file containing source data pertaining to Figure 4F.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Localization of Bodipy-lipids to Cav1-mCh positive for EHD2-BFP.
(A) Cav1-mCh HeLa cells transiently expressing EHD2-BFP were incubated with Bodipy-lipid liposomes. White lines shown in Figure 4A–D indicate the location of the kymograph and the corresponding …
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Figure 4—figure supplement 1—source data 1
Excel file containing source data pertaining to Figure 4—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Kymographs of Cav1-mCh HeLa cells incubated with Bodipy-lipid liposomes.
(A–D) Cav1-mCh HeLa cells were incubated with Bodipy-lipid liposomes as indicated. White lines indicate the location of the kymograph and the corresponding intensity profiles illustrate the …
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Figure 4—figure supplement 2—source data 1
Excel file containing source data pertaining to Figure 4—figure supplement 2A–D.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Bodipy-Chol and Bodipy-SM C12 are sequestered within caveolae.
(A, B) Bodipy fluorescence recovery experiments to study the accumulation of lipids in caveolae. Cav1-mCh HeLa cells transiently expressing EHD2-BFP were incubated with liposomes containing either (A…
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Figure 4—figure supplement 3—source data 1
Excel file containing source data pertaining to Figure 4—figure supplement 3C.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig4-figsupp3-data1-v2.xlsx
Figure 4—video 1
Bodipy-LacCer colocalizes with Cav1-mCh positive structures.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells after incubation with liposomes containing Bodipy-LacCer. The image in Fig. 4A is taken from this movie and corresponds to …
Figure 4—video 2
Bodipy-Chol colocalizes with Cav1-mCh positive structures.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells after incubation with liposomes containing Bodipy-Chol. The image in Fig. S4A is taken from this movie and corresponds to the …
Figure 4—video 3
Bodipy-LacCer accumulates in caveolae.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells transiently expressing EHD2-BFP after incubation with liposomes containing Bodipy-LacCer. The image in Fig. 4B is taken from …
Figure 4—video 4
Bodipy-Chol accumulates in caveolae.
A representative TIRF live cell movie of Dox-induced Cav1-mCh HeLa cells transiently expressing EHD2-BFP after incubation with liposomes containing Bodipy-Chol. The image in Fig. S4B is taken from …
Figure 5 with 2 supplements
Chol accumulation reduces the caveolae diameter in 3T3 adipocytes.
(A) Representative overlays of light microscopy images with corresponding electron micrographs showing localization of caveolae (Cav1-mCh in red) and nuclei (DAPI in cyan) for untreated Cav1-mCh …
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Figure 5—source data 1
Excel file containing source data pertaining to Figure 5D and E.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Correlative electron microscopy of caveolae.
(A) Cav1-mCh HeLa cells were induced with Dox and transiently expressed EHD2-GFP. Light microscopy image showing localization of caveolae (Cav1-mCh) and nuclei (DAPI) within cells (LM, left panel). …
Figure 5—figure supplement 2
Expression of EHD2 and Cav1 is upregulated in 3T3-L1 adipocytes.
(A) Representative immunoblots showing protein expression of EHD2 and Cav1 during 3T3-L1 differentiation. Clathrin HC served as loading control. (B) Representative electron micrographs of 3T3-L1 …
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Figure 5—figure supplement 2—source data 1
Excel file containing source data pertaining to Figure 5—figure supplement 2C.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig5-figsupp2-data1-v2.xlsx
Figure 6
GSLs are internalized to the endosomal system independent of Cav1, while Chol is predominantly trafficked to lipid droplets.
(A) Cav1-mCh HeLa cells expressing Rab5-BFP were incubated with Bodipy-labeled LacCer or Chol for 15 min. Individual channels are shown for selected areas (dotted box). (B) Colocalization of lipids …
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Figure 6—source data 1
Excel file containing source data pertaining to Figure 6B,C,E,H and I.
- https://cdn.elifesciences.org/articles/55038/elife-55038-fig6-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-PTRF (Rabbit polyclonal) | Abcam | Cat # ab48824 RRID:AB_88224 | IF(1:100) |
| Antibody | anti-EHD2 (Rabbit polyclonal) | (Morén et al., 2012) | RRID:AB_2833022 | IF (1:500), WB(1:2000) |
| Antibody | anti-clathrin heavy chain (clone 23; mouse monoclonal) | BD Transduction Laboratories | Cat # 610499 RRID:AB_397865 | WB (1:1000) |
| Antibody | anti-caveolin-1 (Rabbit, polyclonal) | Abcam | Cat # ab2910 RRID:AB_303405 | IF (1:500) WB (1:10 000) |
| Antibody | anti-rabbit IgG secondary antibody coupled to Alexa Fluor 647 (Goat polyclonal) | Thermo Fisher Scientific | Cat # A21246 RRID:AB_2535814 | IF (1:300) |
| Chemical compound, drug | 1,2-dioleoyl-sn -glycero-3-phosphoethanolamine (DOPE) | Avanti Polar Lipids | Cat # 850725 | |
| Chemical compound, drug | 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) | Avanti Polar Lipids | Cat # 890890 | |
| Chemical compound, drug | Bodipy FL C5- Ganglioside GM1 | Thermo Fisher Scientific | Cat # B13950 | |
| Chemical compound, drug | Bodipy FL C5-LacCer | This study (Gretskaya and Bezuglov, 2013) | ||
| Chemical compound, drug | Bodipy FL C5-ceramide | Thermo Fisher Scientific | Cat # D3521 | |
| Chemical compound, drug | Bodipy-Cholesterol | Avanti Polar Lipids | Cat # 810255 | |
| Chemical compound, drug | Bodipy FL C12-spinghomyelin | Thermo Fisher Scientific | Cat # D7711 | |
| Chemical compound, drug | Bodipy FL C5-spinghomyelin | Thermo Fisher Scientific | Cat # D3522 | |
| Chemical compound, drug | Bodipy FL succinimidyl ester (EverFluor FL) | Setareh Biotech | Cat # 7150 | |
| Chemical compound, drug | D-lactosyl-ß1–1'-D-erythro-sphingosine | Avanti Polar Lipids | Cat # 860542 | |
| Chemical compound, drug | D-lactosyl-ß−1,1' N-palmitoyl-D-erythro-sphingosine | Avanti Polar Lipids | Cat # 860576 | |
| Chemical compound, drug | Cholesterol | Sigma Aldrich | Cat # C3045 | |
| Chemical compound, drug | N-Palmitoyl-D-sphingomyelin | Sigma Aldrich | Cat# 91553 | |
| Chemical compound, drug | Sphingomyelinase (SMase) (from Bacillus cereus) | Sigma Aldrich | Cat# S7651 | |
| Chemical compound, drug | Myriocin (from Mycelia sterilia) | Sigma Aldrich | Cat# M1177 | |
| Chemical compound, drug | Paraformaldehyde 16% | Electron Microscopy Sciences | Cat # 15710 | |
| Chemical compound, drug | Paraformaldehyde | Thermo Fisher Scientific | Cat # PA0095 | |
| Chemical compound, drug | Glutaraldehyde 25% | Taab Laboratory Equipment Ltd | Cat # G011/2 | |
| Chemical compound, drug | 3-isobutyl-1-methylxanthine (IBMX) | Sigma Aldrich | Cat # I5879 | |
| Chemical compound, drug | Dexamethasone (Dex) | Sigma Aldrich | Cat # D4902 | |
| Chemical compound, drug | Insulin | Sigma Aldrich | Cat # I0516 | |
| Chemical compound, drug | rosiglitazone | Cayman Chemicals | 71740 | |
| Chemical compound, drug | Lipofectamine2000 | Thermo Fisher Scientific | Cat # 11668019 | |
| Chemical compound, drug | N,N-Diisopropylethylamine | Sigma Aldrich | Cat # D125806 | |
| Chemical compound, drug | N,N-Dimethylformamide, anhydrous | Sigma Aldrich | Cat # 227056 | |
| Chemical compound, drug | Chloroform | VWR | Cat # VWRC22711.324 | |
| Chemical compound, drug | Methanol | Thermo Fisher Scientific | Cat # 15394528 | |
| Chemical compound, drug | Doxycycline hyclate | Sigma Aldrich | Cat # D9891 | |
| Chemical compound, drug | Hygromycin B | Thermo Fisher Scientific | Cat # 10687010 | |
| Chemical compound, drug | Blasticidin S HCl | Thermo Fisher Scientific | Cat # R210-01 | |
| Cell line (Homo-sapiens) | HeLa (cervix) | ATCC | Cat # CRM-CCL-2 RRID:CVCL_0030 | |
| Cell line (M. musculus) | 3T3-L1 (Pre-adipocytes) | ATCC | Cat # ATC-CL-173 RRID:CVCL_0123 | |
| Cell line (Homo-sapiens) | HeLa FlpIn T-Rex Caveolin1-mCherry | This study | ||
| Cell line (Homo-sapiens) | HeLa FlpIn T-Rex EHD2pTagBFP-P2A-Caveolin1-mCherry | This study | ||
| Transfected construct (human) | siRNA to EHD2 (Stealth) | Thermo Fisher Scientific | Cat#1299001 Assay ID HSS121265 | |
| Transfected construct (human) | Scrambled 353 siRNA (Stealth) | Thermo Fisher Scientific | Cat# 12935300 | |
| Transfected construct (human) | siRNA to Caveolin1 (Stealth) | Thermo Fisher Scientific | Cat #1299001 Assay ID HSS141467 | |
| Recombinant DNA reagent | Rab5-TagBFP | (Francis et al., 2015) | under control of PCMV IEpromotor in pTagBFP-N vector | |
| Recombinant DNA reagent | EHD2-TagBFP | This study | under control of PCMV IEpromotor in pTagBFP-N vector | |
| Recombinant DNA reagent | EHD2 I157Q-TagBFP | This study | under control of PCMV IEpromotor in pTagBFP-N vector | |
| Recombinant DNA reagent | EHD2-mCherry | (Hoernke et al., 2017) | under control of PCMV IEpromotor in pmCherry-N1 vector | |
| Recombinant DNA reagent | mCherry-EHD2 | This study | under control of PCMV IEpromotor in pmCherry-C1 vector | |
| Recombinant DNA reagent | EHD2 I157Q-mCherry | This study | under control of PCMV IEpromotor in pmCherry-N1 vector | |
| Recombinant DNA reagent | mCherry-EHD2 I157Q | This study | under control of PCMV IEpromotor in pmCherry-C1 vector | |
| Recombinant DNA reagent | pCDNA/FRT/TO caveolin1-mCherry | This study | under control of PCMV IE promotor | |
| Recombinant DNA reagent | pCDNA/FRT/TO EHD2-pTagBFP-P2A-caveolin1-mCherry | This study | under control of PCMV IE promotor | |
| Software, algorithm | ImageJ/Fiji | Fiji (Schindelin et al., 2012) | RRID:SCR_002285 | http://fiji.sc/ |
| Software, algorithm | Imaris x64 9.1.2 | Bitplane | RRID:SCR_007370 | http://www.bitplane.com/imaris |
| Software, algorithm | Prism 5.0 | GraphPad | RRID:SCR_002798 | https://www.graphpad.com/scientific-software/prism/ |
| Software, algorithm | Photoshop CS6 | Adobe | RRID:SCR_014199 | https://www.adobe.com/se/products/photoshop.html |
| Software, algorithm | Illustrator CS6 | Adobe | RRID:SCR_010279 | https://www.adobe.com/se/products/photoshop.html |
| Software, algorithm | Maps 3.3 | FEI | https://www.fei.com/software/maps/ | |
| Software, algorithm | Zen interface 2.3 | Zeiss | RRID:SCR_013672 | https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html |
| Software, algorithm | Nis Elements 4.3 | Nikon | RRID:SCR_014329 | https://www.nikoninstruments.com/en_EU/Products/Software |
| Other | Aluminum backed silica gel plates (median pore size 60 Å, fluorescent indicator 254 nm) | Fisher Scientific | Cat # 10517771 | |
| Other | Chromatography grade silica gel (0.035–0.070 mm, 60 Å) | Acros Organic | Cat # 240360050 | |
| Other | Formvar TEM grids | Taab Laboratory Equipment Ltd | Cat # F005 | |
| Other | CS-25R17 coverlips (TIRF) | Warner Instruments | Cat # 64–0735 | |
| Other | CS-25R15 coverlips | Warner Instruments | Cat # 64–0715 | |
| Other | Precision cover glasses thickness No. 1.5H | Paul Marienfeld GmbH and Co. KG | Cat # 0117520 | |
| Other | PD-10 columns | GE Healthcare | Cat # 17-0851-0 | |
| Other | MatTek dishes (35 mm dish, high tolerance 1.5) | MatTek Corporation | Cat # P35G-0.170–14 C | |
| Other | HCS LipidTOX Deep Red Neutral Lipid Stain | Thermo Fisher Scientific | Cat # H34477 | |
| Other | Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific | Cat # 41966052 | |
| Other | Opti-MEM I Reduced Serum Medium | Thermo Fisher Scientific | Cat # 31985070 | |
| Other | Dulbecco's Modified Eagle Medium (DMEM), no phenol red | Thermo Fisher Scientific | Cat # 21063029 | |
| Other | Sodium pyruvate | Thermo Fisher Scientific | Cat # 11360039 | |
| Other | Dako Fluorescent Medium | Dako | Cat # S3023 | |
| Other | Alexa Fluor 647 C2 Maleimide | Thermo Fisher Scientific | Cat # A20347 | |
| Other | DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | Thermo Fisher Scientific | Cat # D3571 | |
| Other | Fetal bovine serum | Thermo Fisher Scientific | Cat # 16000044 |
Table 1
Retention times (RT), MRM-transition stages monitored (precursor ion and product ions) and collision energies of analyzed compounds.
| Compounds | MRM transition | RT (min) | Collision energy (V) | |
|---|---|---|---|---|
| Precursor ion | Product ion | |||
| Bodipy-LacCer | 926.5 | 562.4 | 1.48 | 30 |
| LacCer(d18:1/16:0) | 862.6 | 520.5 | 2.84 | 20 |
| LacCer(d18:1/14:0) | 834.6 | 264.3 | 2.8 | 40 |
| SM(d18:1/16:0) | 703.6 | 184.1 | 2.9 | 30 |
| Bodipy-SM C12 | 865.6 | 184.1 | 2.12 | 30 |
Table 2
MRM transitions for labeled and endogenous Chol.
| Compound | Comment | Precursor ion | MS1 resolution | Product ion | MS2 resolution | RT | RT delta min (total) |
|---|---|---|---|---|---|---|---|
| Methyl stearate | IS-std | 298 | Unit | 101.1 | Unit | 5.6 | 2 |
| Chol | Quant | 329 | Unit | 95 | Unit | 7.8 | 2 |
| Chol | Qual | 368 | Unit | 213 | Unit | 7.8 | 2 |
| d7-Chol | Quant | 336 | Unit | 95 | Unit | 7.8 | 2 |
| d7- Chol | Qual | 375 | Unit | 213 | Unit | 7.8 | 2 |
