Supporting Information
Combined Transcriptomics and Chemical-Genetics Reveal Molecular Mode of Action of Valproic acid, an Anticancer Molecule using Budding Yeast Model 1
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Upendarrao Golla, 1Deepthi Joseph, and 1Raghuvir Singh Tomar*
Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Bhopal-462066, India
Contents of Supporting Information Number of pages: 145 Figures: S1-S10 Tables: S1-S6
*Correspondence to be addressed: Raghuvir S. Tomar, Associate Professor, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal–462066, India. Tel.: +91-755- 6692560; Fax: +91-755- 4092392; Email:
[email protected]
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SUPPLEMENTARY METHODS: Cell Viability Assay: The viability of yeast cells after VA treatment was assessed by using methylene blue (vital dye) staining method as described earlier 1. Exponentially growing wild-type (1588-4C) yeast cells were left untreated (control) or treated with VA (2, 4, 6, and 8 mM) for 3 h at 30 o
C. A fraction of cells from each treatment were stained with 3.7% buffered methylene blue solution.
Heat killed (70 oC/15 min) cells were served as a positive control. Cells stained dark blue were considered as metabolically inactive or dead. The staining of yeast cells was recorded by using a LEICA DM500 microscope (installed with LAS EZ V1.7.0 software) at 400X total magnification. Functional Enrichment Analysis of VA Transcriptome: The DEG’s were systematically classified into MIPS (Munich Information Center for Protein Sequences) functional categories using Functional Catalogue Database (FunCatDB; available at http://mips.helmholtzmuenchen.de/funcatDB/)2, and evaluated for enrichment of functional gene clusters using a webbased tool, FunSpec (http://funspec.med.utoronto.ca)3. Moreover, GO clustering and enrichment analysis was performed using Biological Networks Gene Ontology (BiNGO) tool (http://apps.cytoscape.org/apps/bingo)4 and Gene Ontology Enrichment Analysis Software Toolkit (GOEAST, available at http://omicslab.genetics.ac.cn/GOEAST/)5 respectively. Significantly enriched GO categories were represented in the form of an interactive hierarchy using Cytoscape software6. Additionally, functional enrichment analysis of DEG’s was carried out using FunCat classification in FungiFun2 tool (https://elbe.hki-jena.de/fungifun/fungifun.php)7. The transcriptional factors (TF’s) present in DEG’s were identified and ranked (based on % of transcriptome regulation) using YEASTRACT tool (http://www.yeastract.com/)8. Also the enrichment of chromatin features in up-regulated genes in VA transcriptome was analyzed by using ChromatinDB database (http://www.bioinformatics2.wsu.edu/ChromatinDB)9.
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SUPPLEMENTARY FIGURES:
Figure S1:
Figure S1: Acute exposure of Valproic acid (VA) reversibly inhibits growth and didn’t affect the cell viability. (a) Exponentially growing wild-type (1588-4C) yeast cells in SC-liquid media were left untreated (Control) or treated with 2, 4, 6 and 8 mM of VA for 3 h at 30 oC. The cell viability was assessed by staining with 0.3% buffered methylene blue (vital dye) and visualized under light microscope (400X). Untreated and heat-killed yeast cells were served as negative and positive control respectively. (b) Exponentially growing wild-type (1588-4C and BY4741) yeast cells were left untreated (UN) or treated with indicated VA doses and the growth was recorded for 6 h at 30 oC. After VA treatment for 6 h, the cells were washed, resuspended (indicated by dotted vertical line in graph) in fresh SC-liquid media at an equal OD600 of 0.3 and the growth was monitored at a regular interval of time for 20 h using plate reader. The averaged (n=3) OD values were used to construct the growth curves.
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Figure S2:
Figure S2: Validation of VA Transcriptome by RT-qPCR. (a) The table shows the list of genes selected for validation along with their relative fold change and regulation (up or down) in VA transcriptome. These selected genes represent diverse cellular processes including HOG pathway (HOG1), CWI pathway (SPS100, WSC3), heavy metal homeostasis (FRE1), Detoxification (DIT2), Meiosis (DMC1, DIT1, SPS1, SPO13, and SPO20), lipid/fatty acid transport (GIT1), Receptor sensor and morphogenesis (WSC3), Iron ion binding and transport (FRE1 and FET3). (b) RT-qPCR. Total RNA was isolated from exponentially growing cells that were left untreated or treated with VA (6 mM) for 3 h and reverse transcribed to cDNA. The relative expression (fold change) values of selected genes were represented as Mean±SEM (n=3). *p
