Table 1.
Oligonucleotides inserted into the BbsI site of pSpCas9(BB)-2A-GFP.
sgRNA sequences are indicated in bold and preceded by a G nucleotide to improve transcription [29].
Fig 1.
Characterization of the MEF Sgpl1+/+ and Sgpl1-/- cell lines.
(A) Sgpl1+/+ and Sgpl1-/- cells were metabolically labeled with 6 μM pacSph for 4 h. Lipids were extracted and subjected to click reaction with fluorogenic coumarin azide, separated by TLC and exited with UV light. Lipids were identified via clickable standards (S3 Fig). Three replicates are shown for every cell line. (B) Quantification of the fluorescence intensities of Fig 1A. Values are listed in S1 Table. Mean ± SD are shown (n = 3). (C) Samples were treated as above, but were also subjected to alkaline hydrolysis (saponified) or mock treatment. (D) Membrane Lipidome. Lipid class profile of Sgpl1+/+ and Sgpl1-/- cells. Lipid classes are standardized to all lipids measured excluding storage lipids (TAG and CE). GP O- lipids are contained in the sum of each class (e.g. PE O- in PE) and may show overlap with diacyl species containing odd-numbered fatty acids. PE Plasmalogens (PE P-) are displayed as a separate class. A Welch Two Sample t-test was used to estimate the P values: *P < 0.05; **P < 0.01; *** P < 0.001. Error bars correspond to standard deviation (n = 6).
Fig 2.
Characterization of the HeLa ΔSGPL1 cell lines.
(A) HeLa wildtype (H, Lane 1) and HeLa ΔSGPL1 (Δ,Lane 2) cells were labeled with 6 μM pacSph for 6 h. Lipids were extracted and subjected to click reaction with fluorogenic fluorescein, separated by TLC and excited with blue light. Lipids were identified based on comparison to the respective lipid standards (see Fig 2C). Both samples were also subjected to alkaline hydrolysis (Lane 3 and 4). (B) Quantification of the fluorescence intensities of Fig 2C. Mean ± SD (n = 3) are shown (S2 Table). Each timepoint is standardized to 100% of lipids quantified. Data are fitted by Local Polynomial Regression Fitting (LOESS, degree = 2) as curves. (C) HeLa ΔSGPL1 cells were labeled for the time points indicated and treated as in (A). As reference, 50 pmol of each clickable Cer, HexCer, PC and SM (S3 Fig) was loaded as standard.
Fig 3.
Rescue of HeLa ΔSGPL1 cells by exogenous Flag-SGPL1.
HeLa wildtype, HeLa ΔSGPL1 and HeLa ΔSGPL1 transfected with Flag-tagged SGPL1 were labeled with 6 μM pacSph for 4 h. Upper Panel: Proteins precipitated during the lipid extraction were solubilized, analyzed by SDS-PAGE and immunoblotting, using anti-FLAG antibodies (red). Detection of endogenous calnexin with anti-calnexin antibody (CNX, green) was used as a loading control. Lower Panel: Extracted lipids were subjected to click reaction with fluorogenic coumarin azide, separated by TLC and exited with UV light. Lipids were identified via with alkyne lipid standards (S3 Fig).
Fig 4.
Lipidomics analysis of the HeLa and HeLa ΔSGPL1 cell lines.
(A) Class profile, standardized to all lipids measured without storage lipids (TAG and CE). GP O- lipids are contained in the sum of each class (e.g. PE O- in PE) and may show overlap with diacyl species containing odd-numbered fatty acids. PE P-lipids are displayed as a separate class. (B) Sphingoid bases and sphingoid base 1-phosphates standardized to total phosphate (fmol/nmol Pi). Data are shown in S3 Table. A Welch Two Sample t-test was used to estimate the P values: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars correspond to standard deviation (n = 3).
Fig 5.
Proteomic analysis of the HeLa and HeLa ΔSGPL1 cell lines.
Differential expression of proteins in HeLa and HeLa ΔSGPL1 cell lines. (A and C) Proteins only found in one of the cell lines. Protein LFQ (label free quantification) intensities of HeLa (A) or HeLa ΔSGPL1 (C) cells were used to calculated T-test p-values. A list of proteins is provided in S5 and S6 Tables. (B) Volcano plot of differential expression of proteins. Difference of log transformed LFQ (label free quantification) intensities of HeLa cells and HeLa ΔSGPL1 cells (ΔSGPL1 –HeLa). Statistical analysis was carried out with Perseus software. Significance Values are based on permutation based FDR analysis [44]: Proteins scoring p < 0.05 are shown in green, p < 0.01 in red. A list of proteins is provided in the S4 Table.
Fig 6.
Fluorescent labeling of pacSph metabolites to study protein lipid interaction.
Flag-tagged STARD7 (A) and p24 (B) were expressed in HeLa and HeLa ΔSGPL1 cell lines. Cells were metabolic labeled with 5 μM pacSph (STARD7) or 0.5 μM pacSph with or without sphingosine (p24) for 7 h and then UV irradiated to cross-link pacSph metabolites to nearby protein. Protein lysates were subjected to click reaction with Alexa647 azide (shown in red) and the ectopically expressed proteins were immunoprecipitated. After SDS-PAGE and immunoblot with fluorescently labeled secondary antibodies (shown in green), lipid and protein signals were detected in separate channels. Lys, Lysate, IN, input of immunoprecipitation, IP, immunoprecipitated material.