Additional file 1 of Oleic acid from cancer-associated fibroblast promotes cancer cell stemness by stearoyl-CoA desaturase under glucose-deficient condition
Creators: name: Hwang, Sung-Hyun
name: Yang, Yeseul
name: Jung, Jae-Ha
name: Kim, Yongbaek
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Cancer
subject: Computational Biology

description: Additional file 1. Figure S1. Phenotype of CAFs isolated from xenografted tumor masses (A) Staining for CD90 and FAP in H460 cells, NFs and CAFs. (B) Representative image of oil red O staining in a xenografted tumor. Red fluorescence indicates oil red O staining. 4× magnification. Scale bar: 150 μm. (C) Representative histogram for BODIPY staining in H460 cells, CAFs and NFs. The P2 population was established using H460 cells. ***P &lt; 0.001 vs. H460 cells. Data indicate the mean ± SEM (n = 3). (D) Profile of fatty acid contents in supernatant from control and 1 μM CAY10566-treated CAFs. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Data represent the mean ± SEM (n = 3). (E) Transcription levels of ACLY, ACAC, and FASN in H460 cells incubated in complete medium with supernatant of NF or CAF.1 for one day and subsequently treated with CAY10566. *P &lt; 0.05. Data represent the mean ± SEM (n = 4). Figure S2. Effect of glucose deficiency on lipid metabolism and autophagosome. (A) Representative fluorescence image of BODIPY staining in H460 cells incubated with NF or CAF supernatant in complete or glucose-deficient medium for one day. 20× magnification. Scale bar: 50 μm. (B) Assessment of FFA levels in H460 cells incubated with supernatant of NF or CAF in complete or glucose-deficient medium for one day. $$P &lt; 0.01, $$$P &lt; 0.0001 vs. treatment of H460 cells with H460 supernatant. #P &lt; 0.05, ###P &lt; 0.0001 vs. treatment of glucose starved H460 cells with H460 supernatant. **P &lt; 0.01 and ***P &lt; 0.001. Error bars indicate SEM (n = 4). (C) Transcription levels of SREBP-1c and SCD in H460 cells incubated with supernatant of NF or CAF and cultured in complete or glucose-deficient medium for one day. #P &lt; 0.05, ##P &lt; 0.01, ###P &lt; 0.001 vs. treatment of H460 cells with H460 supernatant. $$P &lt; 0.01, $$$P &lt; 0.001 vs. treatment of glucose starved H460 cells with H460 supernatant. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Error bars indicate SEM (n = 3). (D) Transcription levels of SREBP-1c and SCD in H460 cells treated with OA in complete or glucose-deficient medium. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Data indicate the mean ± SEM (n = 4). (E) Representative flow cytometric analysis of Cyto-ID staining in H460 cells treated with OA and CAY10566. The bar graph indicated the mean value. **P &lt; 0.01 and ***P &lt; 0.001. Data represent the mean ± SEM (n = 3). (F) Expression of ATG5, LC3-I, LC3-II, and β-actin in H460 cells treated with OA in complete or glucose-deficient medium. (G) Representative fluorescence image of Oct4, BODPY, and DAPI in H460 cells treatment with OA or not in the presence or absence of glucose. 20× magnification. Scale bar: 200 μm. Figure S3. Characterization of SCD gene-manipulated cells and their side population (SP) (A) Protein expression and (B) transcription level of SCD in control Cas9 (Mock) cells and colony of SCD-KO cells. **P &lt; 0.01 vs. mock cells. Data indicate the mean ± SEM (n = 4). (C) Expression of SCD in parental, control Cas9, SCD-KO, control-pCDH, and SCD-pCDH H460 cells. (D) Profile of fatty acid contents in subclones of H460 cells. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001, Data represents the mean ± SEM (n = 3). (E) Expressions of Atg5 and BECN1 in parental, SCD-KO, and SCD-pCDH H460 cells. (F) Expression of Nanog, Oct4 and β-actin, and (G) staining for CD133 and CD44 in parental, SCD-KO, and SCD-pCDH H460 cells. (H) Representative flow cytometry for SP assay in H460 cells treated with verapamil, parental, SCD-KO, and SCD-pCDH H460 cells. ***P &lt; 0.001. Data indicate the mean ± SEM (n = 3). (I) Representative image for spheroid assay in SCD subclones in the presence or absence of OA. Spheroids over 100 μm in diameter were counted using Image J. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Data represent the mean ± SEM (n = 5). (J) Normalization of phosphor-cofilin and F-actin with cofilin and β-actin. Data represent the mean ± SEM (n = 3). (K) Expression of β-actin, α-tubulin, and Lamin A/C in whole lysate, cytoplasmic, and nuclear of H460 cells. Figure S4. Effects of SCD on mitochondrial function and dynamics machinery. (A) Representative fluorescence image of mitotracker, F-actin, and DAPI in parental, SCD-KO, and SCD-pCDH H460 cells. Arrow indicates co-localized fluorescence of F-actin and mitotracker. 40× magnification. Scale bar: 100 μm. (B) Representative flow cytometry for JC-1 staining in parental, SCD-KO, and SCD-pCDH H460 cells. CCCP was used as a positive control to determine the proportion of red and green regions. ***P &lt; 0.001. Data indicate the mean ± SEM (n = 3). (C) Representative flow cytometry of Rhod-2AM staining in parental, SCD-KO, and SCD-pCDH H460 cells. The P2 population was established using parental H460 cells. ***P &lt; 0.001. Data represents the mean ± SEM (n = 3). (D) Representative fluorescence image of YAP and DAPI in parental and SCD-pCDH cells in presence or absence of etomoxir. 40× magnification. Scale bar: 10 μm. Figure S5. Association of SCD expression in tissues of lung cancer patients (A) Representative IHC staining for SCD in xenografted tumors generated from parental, SCD-KO, and SCD-pCDH cells. 5× and 20× magnification. (B) The mean fluorescence of DAPI and YAP co-localization was measured using ImageJ software. **P &lt; 0.01, and *** P &lt; 0.001. Data indicate the mean ± SEM. (C) Overall survival rates of low- and high-SCD groups in lung adenocarcinoma patients that were available in TCGA datasets. (D) Statistical analysis of DAB intensity is based on quantification of the percentage of SCD+ area in each TMA tissue core using ImageJ software. *P &lt; 0.05, **P &lt; 0.01, and *** P &lt; 0.001. Data indicate the mean ± SEM.