INTEGRATION OF GENERAL AMINO ACID CONTROL AND TOR REGULATORY PATHWAYS IN NITROGEN ASSIMILATION IN YEAST
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1 INTEGRATION OF GENERAL AMINO ACID CONTROL AND TOR REGULATORY PATHWAYS IN NITROGEN ASSIMILATION IN YEAST Kirk A. Staschke 1, Souvik Dey 1, John M. Zaborske 2, Lakshmi Reddy Palam 1, Jeanette N. McClintick 1, Tao Pan 2, Howard J. Edenberg 1, and Ronald C. Wek 1 * 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. 2 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA. SUPPLEMENTAL FIGURES Figure S1. Loss of the GAAC renders cells growth resistant to rapamycin. (A) Wild-type cells, or strains containing the indicated gene mutations, were streaked on YPD agar plates containing 200 nm rapamycin (Rap), 2 mm L-methionine sulfoxamine (MSX), or no stress treatment (YPD). As a control, strains were also grown on synthetic complete medium containing 30 mm 3-amino-triazole (3-AT). (B) A GCN4-lacZ reporter plasmid without uorfs (GCN4- uorfs-lacz) was introduced into wild-type cells (WY837), or cells deleted for GCN2 (WY838) and/or GLN3 (WY840), as indicated in the legend. Cells were treated with 3-AT or rapamycin for 6 hours, and -galactosidase activity was measured from whole cell extracts. The average -galactosidase activity from three independent cultures SEM is shown. Figure S2. Requirements for GCN2, GCN4, and GLN3 for gene expression changes in response to 3-AT or rapamycin treatment. Venn diagrams illustrating the number of genes whose encoded mrnas require GCN2, GCN4, or GLN3, as indicated, for a 2-fold (A) or 2-fold (B) change in expression following 3-AT or rapamycin treatment. The number of transcripts requiring two or all three of these regulatory genes for full induction or repression are indicated inside the colored overlapping regions. Figure S3. Comparative analysis of genes induced by 3-AT or rapamycin treatment. (A) A comparative analysis of genes induced or repressed by 3-AT in this study (RW01) with those in Set A of Natarajan et al., 2001 (5). A Log 10 scatter plot is shown displaying all probe sets representing transcripts which showed a significant change (P 0.05, fdr 0.2) in expression following treatment with 10 mm 3- AT for 1 hour. Transcripts which correlated positively (2103 total) or negatively (206 total) are indicated by black and grey stars, respectively. A trend line is shown in red for those transcripts which displayed a significant positive correlation (Pearson r = 0.87, P < , Spearman r = 0.85, P < ). Transcripts which changed by 2-fold or more (-0.33 Log ) are enclosed by blue dotted lines in upper right and lower left quadrants. (B) The distribution of the yeast transcriptome following treatment with 3-AT (top panel) or rapamycin (bottom panel) is depicted as a histogram indicating the number of genes that were significantly induced or repressed (P 0.05, fdr 0.2). The number of probe sets representing transcripts at each value of Log 2 ratio are plotted. (C) A comparative analysis of genes induced or repressed by rapamycin in this study (RW01) with those in Marion et al., 2004 (14). A Log 10 scatter plot is shown displaying all probe sets representing transcripts which showed a significant change (P 0.05, fdr 0.2) in expression following treatment with 200 nm rapamycin for 1 hour. Transcripts which correlated positively (2390 total) or negatively (371 total) are indicated by black and grey stars, respectively. A trend line is shown in red for those transcripts which displayed a significant positive correlation (Pearson r = 0.87, P < , Spearman r = 0.88, P < ). Transcripts which changed by 2-fold or more (-0.33 Log ) are enclosed by blue dotted lines in upper right and lower left quadrants. (D) A comparative analysis of transcripts induced or repressed following treatment with 3-AT or rapamycin. A Log 2 scatter plot is shown displaying all probe sets representing mrnas which showed a significant change (P 0.05, fdr 0.2) in expression following treatment with 10 mm 3-AT or 200 nm rapamycin for 1 hour. Transcripts which correlated positively or negatively are indicated by black and grey stars, respectively. A trend line is shown in red for those transcripts which displayed a significant
2 positive correlation (Pearson r = 0.73, P < , Spearman r = 0.78, P < ). Transcripts whose levels changed by 2-fold or more (-1.0 Log2 1.0) are enclosed by blue dotted lines in upper right and lower left quadrants. Figure S4. The GAAC pathway is required for expression of ARO9 and ARO10 mrna. Microarray measurements of ARO9 (A) and ARO10 (B) mrna levels were carried out in wild-type (WY798), gcn2 (WY799), gcn4 (WY857), gln3 (WY858), gcn2 gln3 WY859) or gcn4 gln3 WY860) cells, deleted (WY859 and WY860), which were treated with 3-AT, rapamycin, or no treatment (Control), as indicated in the legend. Changes in transcript levels are expressed as fold-change relative to the untreated control in wild-type cells. The mean fluorescence intensity (MFI) values for the ARO9 and ARO10 mrnas are illustrated in Fig. 3. The average fold-change from four independent treatments SEM is shown. Figure S5. qrt-pcr analysis of select transcripts in cells treated with 3-AT or rapamycin. qrt- PCR measurements of select mrnas were carried out in wild-type (WY798), gcn2 (WY799), gcn4 (WY857), and gln3 (WY858) cells, deleted individually or in combination (WY859 and WY860). Cells were treated with 3-AT or rapamycin, or no treatment (Control), as indicated in the legend. Changes in transcript levels are expressed as fold-change relative to the untreated control in wildtype cells. The average fold-change from three to four independent experiments SEM is shown. Figure S6. Induction of GCN4-lacZ activity in the absence of uorfs. (A) -galactosidase activity was measured from wild-type cells (WY837) containing a GCN4-lacZ reporter plasmid without uorfs (GCN4- uorfs-lacz). Cells were grown in synthetic medium containing ammonia as the nitrogen source in the presence (+ AA) or absence (-AA) of all twenty amino acids, or were treated with 10 mm 3- AT (3-AT), 200 nm rapamycin (Rap), or grown in medium containing GABA as the only nitrogen source for 6 hours. The average -galactosidase activity from two independent cultures SEM is shown. (B) -galactosidase activity was measured from wild-type (WY837) or SUI2-S51A (RY287) cells containing a GCN4-lacZ reporter plasmid with uorfs (GCN4-uORFs-lacZ). Cells were grown in synthetic medium as described in panel A. The average -galactosidase activity from three independent cultures SEM is shown.
3 Figure S1. Loss of the GAAC renders cells growth resistant to rapamycin.
4 Figure S2. Requirements for GCN2, GCN4, and GLN3 for changes in gene expression in response to 3-AT or rapamycin treatment.
5 Figure S3. Comparative analysis of genes induced by 3-AT or rapamycin treatment.
6 Figure S3. Comparative analysis of genes induced by 3-AT or rapamycin treatment.
7 Figure S3. Comparative analysis of genes induced by 3-AT or rapamycin treatment.
8 Figure S4. The GAAC pathway is required for expression of ARO9 and ARO10 mrna.
9 Figure S5. qrt-pcr analysis of selected mrnas following treatment with 3-AT or rapamycin.
10 Figure S6. Induction of GCN4-lacZ activity in the absence of uorfs.
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Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific
SUPPLEMENTARY FIGURE LEGEND Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific CD4 + T cells correlates with intracellular CTLA-4 levels. (a) Comparative CTLA-4 levels
Pearson r = P (one-tailed) = n = 9
8F4-Specific Lysis, % 1 UPN1 UPN3 8 UPN7 6 Pearson r =.69 UPN2 UPN5 P (one-tailed) =.192 4 UPN8 n = 9 2 UPN9 UPN4 UPN6 5 1 15 2 25 8 8F4, % Max MFI Supplementary Figure S1. AML samples UPN1-UPN9 show variable
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3021 Supplementary figure 1 Characterisation of TIMPless fibroblasts. a) Relative gene expression of TIMPs1-4 by real time quantitative PCR (RT-qPCR) in WT or ΔTimp fibroblasts (mean ±
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Supplementary Figures and Tables
Supplementary Figures and Tables Supplementary Figure 1. Study design and sample collection. S.japonicum were harvested from C57 mice at 8 time points after infection. Total number of samples for RNA-Seq:
Supplementary Figure S1 Expression of mir-181b in EOC (A) Kaplan-Meier
Supplementary Figure S1 Expression of mir-181b in EOC (A) Kaplan-Meier curves for progression-free survival (PFS) and overall survival (OS) in a cohort of patients (N=52) with stage III primary ovarian
(A) Cells grown in monolayer were fixed and stained for surfactant protein-c (SPC,
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Supplementary Figure 1
Supplementary Figure 1 6 HE-50 HE-116 E-1 HE-108 Supplementary Figure 1. Targeted drug response curves of endometrial cancer cells. Endometrial cancer cell lines were incubated with serial dilutions of
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Supplemental Data. Integrating omics and alternative splicing i reveals insights i into grape response to high temperature
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fl/+ KRas;Atg5 fl/+ KRas;Atg5 fl/fl KRas;Atg5 fl/fl KRas;Atg5 Supplementary Figure 1. Gene set enrichment analyses. (a) (b)
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Supplementary Information. Supplementary Figure 1
Supplementary Information Supplementary Figure 1 1 Supplementary Figure 1. Functional assay of the hcas9-2a-mcherry construct (a) Gene correction of a mutant EGFP reporter cell line mediated by hcas9 or
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
Nature Immunology: doi: /ni.3412
Supplementary Figure 1 Gata1 expression in heamatopoietic stem and progenitor populations. (a) Unsupervised clustering according to 100 top variable genes across single pre-gm cells. The two main cell