Additional file 1 of Fumarate hydratase-deficient renal cell carcinoma cells respond to asparagine by activation of the unfolded protein response and stimulation of the hexosamine biosynthetic pathway
Creators: name: Panarsky, Rony
name: Crooks, Daniel R.
name: Lane, Andrew N.
name: Youfeng Yang
name: Cassel, Teresa A.
name: Fan, Teresa W.-M.
name: W. Marston Linehan
name: Moscow, Jeffrey A.
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Figure S1. Quantitation of Basal Respiration (A), ATP production (B), Glycolysis (E) and Glycolytic Reserve (F) of UOK262/UOK262WT cells under different treatment conditions as measured by XF96 Extracellular Flux Analyzer (C-D and G-H)). 24 x 103 cells/well were seeded 24 h prior to the beginning of the experiment. Basal levels of OCR and proton production were first quantified over approximately 20–25 minutes followed by addition specific inhibitors according to the manufacturer protocol. Three to five independent experimental repeats were carried out. Statistical analysis was performed to compare the untreated versus treated samples using one-way ANOVA test following by unpaired, two-tailed t-tests (GraphPad Prism v. 8). (*)P &lt; 0.05; (**)P &lt; 0.01; (***)P &lt; 0.0001. The significance between treatment groups shown as (#)P &lt; 0.05. Figure S2. (A) Representative 16-pan slide of PathScan Akt Signaling Antibody Array kit performed on UOK262/UOK262WT cells at 3 h and 48 h of incubation in the presence of 100 μM Asn, 2 mM Gln, both amino acids and untreated controls. Fluorescent readout acquisition obtained using the Odyssey Imaging System. Each horizontal pair of dots represents a specific phosphorylated element of the Akt pathway. Three independent experimental repeats were carried out. (B) Change in phosphorylation pattern of pS6RP after different treatments of UOK62/UOK262WT cells after 3 h (top) and 48 h (bottom) of incubation obtained through PathScan antibody array kit. Quantitation performed using ImageJ software. (C) Western Blot analysis of phosphorylation pattern of mTOR, S6 kinase, S6 ribosomal protein, and 4E-BP1 proteins after 48 h of treatments. The UOK262/UOK262WT cells treated with 100 μM Asn, 2 mM Gln or both for 48 h along with untreated control following by cell homogenization. For all Western Blot experiments 20 μg of total protein loaded at each well, unless stated otherwise. Actin used as a loading control. Statistical analysis was performed to compare the untreated versus treated samples using one-way ANOVA test following by unpaired, two-tailed t-tests (GraphPad Prism v. 8). (*)P &lt; 0.05; (**)P &lt; 0.01; (***)P &lt; 0.0001. The significance between treatment groups shown as (#)P &lt; 0.05. Figure S3. Cytotoxicity curves for a Notch signaling inhibitor Fli06 (A), an autophagy inhibitor chloroquine (B), an UPR stress inducer tunicamycin (C) and a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (D) as measured on UOK262/UOK262WT cells under different treatment conditions after 96 h of incubation. The curves represent the percentage of the untreated control. Five independent experimental repeats were carried out. Figure S4. HSQC analysis of UOK262 cells treated with 13C15N Gln + 12C14N Asn. Spectra were recorded as described in the methods. Top KO cells, bottom wt cells. The 1H{13C} HSQC spectrum selects protons attached directly to 13C only. Gln is taken up by the cells and converted to Glu, GSH and fumarate. The free induction decays were multiplied by a 4-Hz line broadening exponential prior to fourier transformation. Table S1. List of treatment unique genes produced by comparison analysis of mRNA-Seq data. Four groups of genes generated based on the gene expression pattern (upregulated and downregulated) affected by incubation with either Asn and Gln or both amino acids.