Journal: eLife
Article Title: Chemotherapy resistance due to epithelial-to-mesenchymal transition is caused by abnormal lipid metabolic balance
doi: 10.7554/eLife.104374
Figure Lengend Snippet: ( A ) Comparison of sphingomyelin (SM) content relative to phospholipid content ( n =3, Student’s t -test). ( B ) Comparison of cholesterol (Chol)/SM ratio between EpH4 and EpH4-Snail cells ( n =3, Student’s t -test). ( C ) Fatty acid composition of SM determined using LC-MS. All peaks corresponding to d18:1-even fatty acid-SMs were included in the analysis and each peak value is expressed as a percentage. n.d., peak not detected ( n =3, Student’s t -test). ( D ) Pie charts of SM chain length profile from (C). SMs were classified into long-chain fatty acid (LCFA, orange) (≤20) and very long-chain fatty acid (VLCFA, purple) (>20) SMs. ( E ) Chain length specificity of Elovls and CerSs responsible for fatty acid profile of sphingolipids reported ( ; ). Elovls catalyze a rate-limiting step of fatty acid elongation cycle, and CerSs catalyze N- acylation of sphingoid bases to produce ceramides. ( F ) Comparison of transcription levels of Elovl s and CerS s. Notable decreases in expressions of Elovl7 and CerS3 in EpH4-Snail cells are indicated by asterisks. ( G ) Quantification of transcription levels of Elovl7 and CerS3 relative to that of ZO-1 , whose expression does not change between EpH4 and EpH4-Snail ( n =3, Student’s t -test). ( H ) Effect of supplementation of C22:0 ceramide (Cer22:0) on ABCA1 expression. EpH4-Snail cells were treated with 10 μM ceramide or 0.4% vehicle (dodecane/ethanol = 2/98) for 24 h, then whole-cell lysates were analyzed by immunoblot ( n =3, Student’s t -test). ( I ) Immunoblot analyses of whole-cell lysates of mouse epithelial (MTD-1A, CSG) and fibroblast (L-929, NIH3T3) cells. ( J ) Comparison of Chol/SM ratio of EpH4, EpH4-Snail, and normal fibroblasts ( n =3, Dunnett’s test). ( K ) Comparison of Chol/SM ratio between TE-15 and TE-8 ( n =3, Student’s t -test). ( L ) Scatter plot of Chol content against SM content used in (B), (H), and (I). Dashed lines indicate Chol/SM = 1.0 (blue) and 1.5 (red). The region of Chol/SM >1.5 is shown with red background. ( M ) Possible mechanism of induction of ABCA1 expression and lipid droplet enlargement through decrease in VLCFA-SM. In EpH4 (epithelial) cells, a certain amount of cholesterol is sequestered through interaction with SM, organizing ordered plasma membrane (PM). In EpH4-Snail (hybrid E/M) cells, the lower level of VLCFA-SM biosynthesis leads to a decrease in plasma membrane SM content, resulting in membrane disorder. The increase in SM-unbound form of cholesterol in PM induces its internalization and activation of cellular cholesterol sensors, resulting in lipid droplet enlargement and enhanced ABCA1 expression. Figure 3—source data 1. Original raw data for the RT-PCR and immunoblot images shown in . Figure 3—source data 2. Labeled uncropped gel and full blot images for the RT-PCR and immunoblot images shown in .
Article Snippet: The following primary antibodies were used for immunoblotting (IB), immunofluorescence microscopy (IF), and immunohistochemistry (IHC): rabbit anti-ABCA1 mAb (96292S, IB, IF), rabbit anti-Snail mAb (3879S, IB), rabbit anti-Akt mAb (4691S, IB), rabbit anti-phospho-Akt (T308) mAb (2965S, IB), and rabbit anti-phospho-FoxO3a (S253) pAb (9466S, IB) from Cell Signaling Technology; rabbit anti-ABCA1 pAb (NB400-105, IHC) from Novus Biologicals; rat anti-E-cadherin mAb (ECCD-2, IB, IF) from TaKaRa; mouse anti-cytokeratin 18 mAb (LS- C84878 , IF) from LS Bio; goat anti-LXR alpha + LXR beta pAb (ab24362, IF) from Abcam; and mouse anti-α-tubulin mAb (12G10, IB) produced in house.
Techniques: Comparison, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Clinical Proteomics, Membrane, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Labeling
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![( A ) Enhancement of growth inhibitory effect of NC on human renal carcinoma cells by co-treatment with an <t>ABCA1</t> inhibitor cyclosporin A (CsA). Cells were grown with or without 20 μM NC and 10 μM CsA for 24 h, and cell number was determined with CCK-8. Fold change in cell number by NC was shown as relative to controls without NC. Results are shown as mean of at least four independent experiments ± SD (Tukey–Kramer’s multiple comparison test). n = 4 (KMRC-3,786-O,A498), 5 (Caki-1) or 6 (TUHR14TKB). ( B–E ) Immunoblot analysis of whole-cell lysate of human renal carcinoma cell lines. A representative result from three independent experiments is shown ( B ). Quantitative analyses of Snail ( C ) and ABCA1 ( D ) expression are presented. A scatter plot illustrating the correlation between ABCA1 and Snail expression levels is also shown ( E ). Quantitative data are shown as mean of three independent experiments ± SD (Tukey–Kramer’s test). ( F ) Comparison of ABCA1 expression between normal tissue and primary tumor of indicated subtypes of renal cancers analyzed using UCSC Xena ( https://xenabrowser.net/ ). Bars indicate means (Welch’s t- test). ( G ) Immunohistochemistry of a surgically extracted renal tissue from a patient with Fuhrman grade 3 primary ccRCC, indicating upregulation of ABCA1 in the lesion site. The ABCA1 staining was verified by the accumulation of known signals in renal tubules and glomeruli in normal tissue . Scale bars, 250 μm (left), 50 μm (right). ( H ) Quantification of ABCA1 signal from surgically extracted renal tissues from three patients for each Fuhrman grade (Tukey–Kramer’s test; see also ). ( I ) Phase-contrast images of EpH4 wild-type cells (top) and Snail-overexpression cells (bottom, EpH4-Snail). Scale bars, 50 μm. ( J ) Immunofluorescence images of EpH4 and EpH4-Snail cells. The cells were fixed. Scale bars, 20 μm. ( K ) Acquisition of NC resistance by exogenous expression of Snail in EpH4 cells and enhancement of 20 μM NC effect on EpH4-Snail cells by co-treatment with 10 μM CsA. Data are presented as in ( A ) ( n = 3, Tukey–Kramer’s test). ( L ) Immunoblot analyses of whole-cell lysates of EpH4, EpH4-Snail, E-cadherin KO, and α-catenin KO EpH4 cells. Quantitative analyses of ABCA1 expression is also shown ( n = 3, Dunnett”s test). ( M ) Immunofluorescence images of EpH4 and EpH4-Snail cells. Cells were fixed with MeOH. Scale bars, 20 μm. ( N, O ) Immunoblot analyses of whole-cell lysates of MDCK II cells ( N ) expressing KRAS G12V treated with 5 μg/ml TGFβ for 0, 3, and 8 days and human esophageal carcinoma cell lines TE-15 and TE-8 ( O ). Graphs on the bottom show analyses from three biological replicates (Tukey–Kramer’s test [ N ] and Student’s t -test [ O ]). Phase-contrast images are also shown on the top ( O ). Scale bars, 50 μm. Figure 1—source data 1. Original raw data for the immunoblot images shown in . Figure 1—source data 2. Labeled full blot images for the immunoblot images shown in .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5503/pmc12795503/pmc12795503__elife-104374-fig1.jpg)
