Additional file 2 of Serine synthesis and catabolism in starved lung cancer and primary bronchial epithelial cells
Creators: name: Haitzmann, Theresa
name: Schindlmaier, Katharina
name: Frech, Tobias
name: Mondal, Ayusi
name: Bubalo, Visnja
name: Konrad, Barbara
name: Bluemel, Gabriele
name: Stiegler, Philipp
name: Lackner, Stefanie
name: Hrzenjak, Andelko
name: Eichmann, Thomas
name: Köfeler, Harald C.
name: Leithner, Katharina
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology

description: Additional file 2: Supplementary Figure S1. Overall survival in lung adenocarcinoma (n=491) and squamous cell carcinoma patients (n=471) from the TCGA dataset expressing low or high levels of SSP or one-carbon metabolism enzymes. Median values were used as a cut-off. Survival analysis was performed using Log-rank test. Supplementary Figure S2. Cell counts and viability under starvation conditions. A Viable cell numbers and B the percentage of viable (calcein positive) cells after treatment with the respective starvation media for 36 hours. Results are mean +/- SEM from four independent experiments. Group comparisons were performed by Two-way ANOVA with Tukey post-hoc analysis. B As a positive cell death control, cells treated with serum-free medium lacking glucose, glutamine, serine and glycine was included. Viability was compared to the respective 10 mM glucose, high serine/glycine conditions using One-way ANOVA with Dunnett post-hoc analysis.*P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; n.s., not significant; # versus high glucose; Ser/Gly, serine/glycine. Supplementary Figure S3. SSP gene expression in lung cancer and normal lung epithelial cells under starvation treatments. A mRNA levels of SSP and one-carbon metabolism genes in two PHGDH-positive cell lines, A549 and H1299 and normal bronchial epithelial cells (BEC) treated in medium containing high glucose and serine/glycine levels for 48 hours. Results are shown as mean +/- SEM from four (A549, H1299) or three (BEC) independent experiments; Group comparisons were performed by One-way ANOVA with Dunnett post-hoc analysis versus BEC for each gene.***P &lt; 0.001. B-D Representative PHGDH and PSAT1 immunoblots and quantifications of blots from four (A549) or three (H1299 cells or BEC cells) experiments cultured in medium containing high (H) or low (L) serine/glycine, and high (H) or low (L) glucose for 48 2 hours (with medium replacement after 24 hours), with or without variations in cystine (high or low, only A549). β-actin was used as a loading control. Data were normalized to β-actin and to nonstarved cells. Group comparisons were performed by two-sided Student’s t-test or one-group Student’s t-test as applicable. Supplementary Figure S4. Serine and glycine abundance in starvation and non-starvation media and serine and glycine enrichment from 13C6-glucose in H460 cells. 13C6-glucose was administered in high glucose medium containing high or low serine/glycine concentrations for 24 hours. A Serine and glycine isotopologue abundance normalized to total protein and internal standard corresponding to Fig. 3. B Serine and glycine enrichment in H460 cells. Fractional enrichments and normalized fractional enrichments are shown. Results are mean +/- SEM from three independent experiments. Group comparisons were performed by A Two-way ANOVA with Tukey post-hoc analysis on total abundance or B two-sided Student’s t-test. *P &lt; 0.05; **P &lt; 0.01;***P &lt; 0.001; n.s., not significant; Ser/Gly, serine/glycine. Supplementary Figure S5. Isotopologue enrichment in TCA cycle and glycolysis intermediates derived from 13C6-glucose. 13C6-glucose was administered in a high or low concentration medium containing high or low serine/glycine concentrations for 24 hours. Isotopologue enrichment (% of total) is shown as mean +/- SEM from four (A549, H1299) or three (BEC) independent experiments. The most informative isotopologue (##) was used for group comparisons using Two-Way ANOVA with Tukey post-hoc analysis. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.0, n.s., not significant; # versus high glucose; Ser/Gly, serine/glycine. Supplementary Figure S6. Metabolite abundance of TCA cycle and glycolysis intermediates derived from 13C6-glucose. Total metabolite abundance normalized to total protein and internal standard from experiments shown in Supplementary Figure S5. Data are 3 shown as mean +/- SEM from four (A549, H1299) or three (BEC) independent experiments. Group comparisons were performed using Two-Way ANOVA with Tukey post-hoc analysis. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; n.s., not significant; # versus high glucose; Ser/Gly, serine/glycine. Supplementary Figure S7. Release of lactate, serine and glycine. 13C6-glucose was administered in a high or low concentration medium containing high or low serine/glycine concentrations for 24 hours in A A549 and B H1299 cells. Isotopologue enrichment in medium metabolites, the amount of de novo produced and released (fully 13C-labeled) metabolites normalized to total protein and total metabolite levels in the medium supernatants are shown as mean +/- SEM from four independent experiments. Statistical analysis (Two-Way ANOVA with Tukey post-hoc analysis). For enrichment data, statistical analysis was done for the most informative isotopologue (##). *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; n.s., not significant; # versus high glucose; Ser/Gly, serine/glycine. Supplementary Figure S8. Isotopologue enrichment in TCA cycle and glycolysis intermediates derived from 13C5-glutamine. 13C5-glutamine was administered in a high or low concentration medium containing high or low serine/glycine concentrations for 24 hours. A Serine M+3 to PEP M+3 ratio from 13C5-glutamine, reflecting serine de novo synthesis from gluconeogenesis. Group comparisons were performed using Students’s t-test. ***P &lt; 0.001. B TCA cycle and glycolytic intermediate enrichments. A,B Data are shown as mean +/- SEM from four (A549, H1299) or three (BEC) independent experiments. Ser/Gly, serine/glycine. Supplementary Figure S9. Isotopologue abundance of TCA cycle and glycolysis intermediates derived from 13C5-glutamine. Normalized isotopologue abundances from experiments shown in Supplementary Fig. S8. Data are shown as mean +/- SEM from four (A549, H1299) or three (BEC) independent experiments. Group comparisons were performed using Two Way ANOVA with Tukey post-hoc analysis. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; n.s., not significant; # versus high glucose; Ser/Gly, serine/glycine. Supplementary Figure S10. Impact of exogenous cystine on labeling of cellular intermediates from 13C5-glutamine. 13C5-glutamine was administered in high or low glucose medium containing high or low serine/glycine in combination with high or low cysteine concentrations. Fractional enrichment of gluconeogenesis and SSP intermediates is shown as mean +/- SEM from three independent experiments.