Additional file 1 of Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis
Creators: name: Hung, Yu-Wen
name: Ouyang, Ching
name: Ping, Xiaoli
name: Qi, Yue
name: Wang, Yi-Chang
name: Kung, Hsing-Jien
name: Ann, David K.
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Figure S1. The ArgS treatment leads to a reduction in global O-GlcNAcylation levels and affects the transcription of HBP and O-GlcNAc recycling enzyme genes.Immunoblot analysis of OGA and OGT in the whole cell extracts from BT-549 cells cultured in the -Gln or -Glc m4dium for 24 h. One representative immunoblotis shown. The relative OGA and OGT protein levels are determined after normalizing against the densitometric signal intensity of the OGA or OGT in the control group, which was set as 1 after normalization with actin or H3 signal.A schematic overview of HBP. The HBP enzymesand metabolitesare depicted.Gene expression of the indicated genes in BT-549 cells after incubation in full medium for 48 h or -Arg medium for 24 and 48 h, respectively. The results of the qRT-PCR analysis of the mRNA encoding HBP enzymes shown in the left panel in BT-549 cells after ArgS treatment for 24 and 48 h, respectively. The qRT-PCR analyses were performed using gene-specific primer pairs, and the 2–∆∆Ct method was used to analyze the results. ΔΔCT = ΔCT− ΔCT.The Staudinger ligation was performed in protein lysates where O-GlcNAz-modified proteins were conjugated with phosphine-PEG3-biotin. The O-GlcNAz-modified proteins were then pulled-down using streptavidin.A heat map shows the abundance ratioof 2054 quantified O-GlcNAz-modified proteins. The scale of the heat map is limited to; n=4.GlcNAz-labeled proteins in BT-549 cells were pulled down and subjected to immunoblot analyses. BT-549 cells were maintained in -Gln and -Glc medium supplemented with GlcNAzfor 48 h prior to cell harvest. O-GlcNaz-modified proteins were collected, followed by immunoblot analysis using an anti-eIF2α antibody to determine the endogenous eIF2α O-GlcNAcylation levels.Data are shown as mean ± s.e.m.; *: p&lt;0.05; **: p&lt;0.01; ***: p&lt;0.001; One-Way ANOVA. Figure S2. Antioxidant gene expression analyses in breast carcinoma samples.Immunoblots of HO-1 in BT-549 and MDA-MB-2321 cells subjected to ArgS or ADI-PEG20treatment for 24 and 48 h; n=3.Immunoblot analysis of HO-1 in BT-549 cells subjected to ArgS for 24 h, followed by Arg recovery for 6, 12, and 24 h. One representative immunoblotis shown.Antioxidant gene HMOX1, CISD2, SOD1, and GSR expression in the normal, tumor and metastatic tissues collected from breast cancer patients were examined.Pancancer analysis of HMOX1 expression across normaland tumor tissues. 17 cancer types in TCGA Pan-Cancer study having &gt;5 normal samples were presented as indicated. Standard boxplots were applied to visualize the log2-transformed HMOX1 expression levelsand the number of samples was labeled at the bottom.HMOX1 expressionin breast tumors across different molecular subtypes and adjacent normal tissues. The number of samples was labeled at the bottom.*: p&lt;0.05; **: p&lt;0.01; ***: p&lt;0.001; N.S.: p&gt;0.05; Wilcoxon tests; p-values were adjusted for multiple comparison using Bonferroni method with the normal samples set as the reference group.Kaplan-Meier survival curves comparing the HO-1 protein levels and overall survivalrates of breast cancer patients. 58 breast tumor samples expressing detectable/no detectable HO-1 in the Tang database were applied to the comparison using log-rank test. Figure S3. HO-1 expression enhances cell recovery from ArgS.Immunoblot analysis of HO1 in different breast cancer cell lines. The whole cell lysates were collected from the different cell lines grown in complete medium.Immunoblot analysis of HO-1 in BT-549 cellstransfected with siHMOX1or treated with ZnPPIX, and in MDA-MB-231 cellsstably overexpressing HO-1 or treated with COPPIX.Measurement of ROS levels in BT-549 and MDA-MB-231 cells subjected to ArgS for 48 h. ROS levels were assessed using DCF-DA oxidation; n=3.Comparison of relative cell viability in BT-549 and MDA-MB-231 cells subjected to ArgS for 24 and 48 h. Assessment of relative cell recovery of MDA-MB-231 cells after treatment with ADI-PEG20for 24 h. The ADIPEG20 treatment was terminated after 24 h; n=3. Comparison of cell recovery in MDA-MB-231 cells overexpressing HO-1 after 24-h treatment with ADI-PEG20; n=3. The ADI-PEG20 treatmentwas terminated by replacing the medium with complete medium. Cell recovery was monitored daily from day 0 to day 3using ACP assays.Assessment of cell migration of MDA-MB-231 cells subjected to ArgS for 24 h; n=3. Cell migration was determined as the percentage of confluence within the gap area.Data are shown as mean ± s.e.m.; *: p&lt;0.05; **: p&lt;0.001; ***: p&lt;0.001; determined by one-way ANOVAor Two-Way ANOVA followed by Tukey's multiple comparison test. Figure S4. eIF2α S51 phosphorylation does not decrease HO-1 expression during ArgS stress.Immunoblot analysis of HO-1 protein abundance in BT-549 cells with defective eIF2α phosphorylation. The effect of defective eIF2α phosphorylation on HO-1 expression was analyzed through the following treatments: 24 h ArgS with tauroursodeoxycholic acid, ISRIB, and phospho-mutant eIF2α. Furthermore, the expression of HO-1 in BT-549 cells stably overexpressing wild-type or O-GlcN-mut or phosphomut eIF2α was analyzed after 24 h of ArgS treatment. One representative immunoblotis shown, with H3 serving as a loading control. The results are shown with parental BT-549 cells serving as a negative control for eIF2α stable expression. The cells were transfected with si-eIF2αor siCtrlfor 48 h prior to ArgS treatment. The relative level of HO-1 proteinwas determined by comparing the densitometric tracing of the HO-1 signal in each experimental condition to the reference HO-1 signal, after normalization with H3. A value greater than 1 indicates an increase in abundance relative to the control, while a value less than 1 indicates a decrease in abundance. Data are presented as mean ± s.e.m.; *: p&lt;0.05; **: p&lt;0.01; ***: p&lt;0.001; Two-Way ANOVA followed by Tukey’s multiple comparison test. Figure S5. HO-1 IHC staining in BT-549 xenografted tumor.Representative IHC staining shows the levels of HO-1 protein in two xenografted tumors harvested from mice that were fed a control dietand -Arg diet.Immunoblot analysis of HO-1 expression in BT-549 cells that were overexpressing a phosphomimickingform of eIF2α. Cells were transfected with FLAG-tagged wild-type or S51D eIF2α expression constructs and then subjected to ArgS. The black arrowheads indicate endogenous eIF2α, and the white arrowheads indicate FLAG-tagged eIF2α. The relative level of HO-1 protein was determined by comparing the densitometric HO-1 signal in the experimental conditions to the reference HO-1. The values of reference HO-1 were set to 1 after normalization with H3, which was used as a loading control.ROS levels in BT-549 cells that were stably expressing wild-type, phosphomut, O-GlcN-mut, or quadruple-mut eIF2α after being subjected to ArgS for 48 h. The ROS level was quantified with CellROX™ via flow cytometry, and the relative oxidized CellROX level is shown after normalization with the value of the control, which was set to 1. The data are shown as mean ± s.e.m.; ns: non-significant, *: p&lt;0.05; **: p&lt;0.01; ***: p&lt;0.001. The analysis was performed using a Two-Way ANOVA followed by Tukey's multiple comparison test. Figure S6. Overall summary. Our data suggest that O-GlcNAcylation of eIF2α is the primary mechanism for downregulating HO-1 protein translation during ArgS. The downregulation of HO- 1 protein leads to an increase in ROS levels and a reduction in cell recovery from ArgS and migratory ability. ArgS induces O-GlcNAcylation on eIF2α, which is considered a stress response to the treatment. The inhibitory effect of increased eIF2α O-GlcNAcylation on antioxidant protein translation reveals a novel mechanism by which ArgS may serve as a potential anti-cancer treatment.