Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables
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1 Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables
2 Supplementary Figure 1: (A), HCT116 IDH1-WT and IDH1-R132H cells were treated with increasing concentrations of ABT199. After 72h of treatment cellular viability was determined via MTT assay. Non-linear regression analysis was performed to determine IC 50 -values. Data are presented as mean and SD, n=3. (B), SF188 pediatric glioblastoma cells were treated for 48h with solvent, ABT199 and 2-HG as indicated. Staining for annexin V/propidium iodide was performed prior to flowcytometric analysis. Right lower quadrant represents early apoptotic cells. Right upper quadrant represents late apoptotic or necrotic cells. Left upper quadrant represents necrotic cells. (C-D), U251 and LN229 cells were treated with vehicle, ABT199, 2-HG or the combination for 48h. Subsequently, cells were stained with annexin V and propidium iodide and analyzed by flow cytometry. 1
3 Supplementary Figure 2: (A), T98G glioblastoma cells were retrovirally transduced with IDH1-WT or IDH1-R132H and expression was induced with doxycycline prior to treatment with solvent or ABT263 as indicated for 48h. Staining with Propidium iodide was performed and the fraction of viable cells (100%- SubG1 cells) was determined by flow cytometry. Column, mean. Error bar, SD, n=3. (B), HCT116 IDH1-WT and IDH1-R132H cells were treated with solvent or ABT263 prior to staining with Propidium iodide and flow cytometric analysis. Column: mean of the fraction of viable cells. Error bar: SD, n=3. (C), T98G IDH1-WT/IDH1-R132H cells were treated with ABT263 at indicated concentrations for 7h. Whole cell extracts were collected and Western blot analysis was performed for caspase 9 (CP9), cleaved caspase 3 (ccp3), PARP and Actin. FL: full length form. CF: cleaved fragment. (D), HCT116 IDH1-WT/IDH1-R132H colorectal cancer cells were treated with ABT263 at indicated concentrations for 7h. Whole cell extracts were collected and Western blot analysis was performed for caspase 9 (CP9), cleaved caspase 3 (ccp3), PARP. Vinculin served as a loading control. FL: full length form. CF: cleaved fragment. 2
4 Supplementary Figure 3: (A-B), SF188 (A) and T98G (B) glioblastoma cells were treated for 48h with solvent, ABT263 and 2-HG as indicated. Staining for annexin V/propidium iodide was performed prior to flowcytometric analysis. Right lower quadrant represents early apoptotic cells. Right upper quadrant represents late apoptotic or necrotic cells. Left upper quadrant represents necrotic cells. (C), SF188 pediatric glioma cells were treated for 48h with ABT263 and 2-HG as indicated prior to staining for Propidium iodide and flowcytometric analysis. Representative flow plots are displayed. 3
5 Supplementary Figure 4: (A), HCT116 IDH1-WT and HCT116 IDH1-R132H cells were treated with increasing concentrations of TRAIL. After 72h of treatment cellular viability was determined via MTT assay. Data are presented as mean and SD, n=3. (B-C), IDH1-mutated and IDH1-WT HCT116 and U251 4
6 glioma cells were treated for 72h with increasing concentrations of TRAIL. Staining for Propidium iodide was performed prior to flowcytometric analysis. Column: mean. Error bar: SD, n=3. (D-E), Representative flow plots of apoptotic HCT116 and U251 cells treated as described for (B-C). (F- G), U87MG IDH1 wild-type or IDH1 R132H mutated cells were treated with increasing concentrations of Etoposide or Paclitaxel and analyzed for cellular viability by CellTiter Glo assay. Data are presented as mean and SD, n=3. 5
7 Supplementary Figure 5: (A), Murine proneural glioblastoma cells, harboring IDH1 wild-type or IDH1 R132H, were analyzed for the expression of Mcl-1, BIM, Bcl-2, Bcl-xL and Actin by Western blot analysis either under low or high glucose conditions. (B), Murine proneural glioblastoma cells, harboring IDH1 wild-type or IDH1 R132H, were analyzed for the expression of mutant IDH1. (C), Scatter plot demonstrating Bcl-2 and Bcl-xL protein expression among IDH1-WT (n=5) and IDH1-R132H (n=6) expressing anaplastic astrocytoma samples (from Figure 3A). Densitometric analysis was 6
8 performed using imagej 1.47v ( Pixel density was normalized to the respective Actin control. (D), Representative flow plots of LN229 cells that were treated with n.t.- sirna or Noxa-siRNA in the presence or absence of 0.5μM ABT263 and 1mM 2-HG for 48h and stained with propidium iodide to determine the fraction of apoptotic cells (subg1 fraction). (E), Representative flow plots of LN229 cells that were treated with n.t.-sirna or Bak-siRNA in the presence or absence of 0.3μM ABT263 and 1mM 2-HG for 48h and stained with propidium iodide to determine the fraction of apoptotic cells (subg1 fraction). (F), U87MG glioblastoma cells were treated for 24h with 2-HG as indicated. Total RNA was collected and isolated prior to performing real-time rtpcr for Mcl-1 mrna. Column: mean. Error bar: SD, n=3. 7
9 Supplementary Figure 6: (A), Wild-type and IDH1 R132H-mutated glioblastoma cells were submitted for transcriptome analysis and subsequently analyzed by GSEA. Shown are GSEA plots with the respective statistical analysis. (B), IDH1 wild-type or IDH1 R132H cells were treated as indicated with solvent, 2-DG or Oligomycin and analyzed for relative ATP content. Column: mean. Error bar: SD, n=3-4. ** indicates a p-value of less than (Student s t-test). (C), U87MG glioblastoma cells were treated with 1 μm ABT263, 5 mm 2-DG or the combination prior to analysis for cellular viability after 48h. Column: mean. Error bar: SD, n=6. ** indicates a p-value of less than 0.01 (Student s t-test). (D), Whole cell extracts were collected from NCH644 IDH1 WT and IDH1 R132H glioma stem-like cells prior to capillary electrophoresis for pampk und AMPK. Vinculin served as a loading control. 8
10 Supplementary Figure 7: (A-C), U87MG glioblastoma cells were treated with 2-HG (1h, 7h or 24h) as indicated and analyzed for oxygen consumption rate (OCR) on a Seahorse XFp Flux analyzer in accordance with the instructions by the manufacturer (Mitochondrial Stress Kit). Data presented as mean and SD, n=3. 9
11 Supplementary Figure 8: (A), U87MG glioblastoma cells were treated for 24h with 2-HG as indicated and subsequently analyzed by capillary electrophoresis for the expression of p-mtor, mtor, p-s6, S6 and Actin. (B), U87MG cells were treated for 5h with 2-HG (1mM) in the presence or absence of the pancaspase inhibitor zvad.fmk (20μM) or the proteasome inhibitor MG-132 (10μM). Whole cell extracts were collected prior to performing Western blot analysis for Mcl-1 and Survivin. Actin served as control for equal loading. 10
12 Glioma stem like cell IC 50 ABT263 (μm) NCH NCH421K 1.83 GS BT NCH Supplementary Table 1: IC50-values for ABT263 in IDH1-WT and IDH1-R132H glioma stemlike cells. Treatment with ABT263 results in a more pronounced response towards ABT263 among IDH1-mutated glioma stem-like cells. NCH644 (IDH1-WT), NCH412K (IDH1-WT), GS9-6 (IDH1-WT), BT-142 (IDH1-R132H) and NCH612 (IDH1-R132H) cells were treated for 72h with increasing concentrations of ABT263 prior to determining IC 50 -values based on CellTiter-Glo assay. U87MG NCH644 GBM12 2-HG (mm) ABT263 (μm) CI 2-HG (mm) ABT263 (μm) CI 2-HG (mm) ABT263 (μm) CI Supplementary Table 2: Combination index-values for a combined treatment with 2-HG and ABT263. ABT263 treatment yields a synergistic antiproliferative effect in the presence of 2-HG. The CompuSyn software (ComboSyn, Inc., Paramus, NJ) was used for the drug-drug interaction analysis including the calculation of the combination index (CI). A CI<1 was considered as synergistic, a CI=1 as additive and a CI>1 as antagonistic. 11
13 Gene Forward sequence Reverse sequence Mcl 1 CCA AGA AAG CTG CAT CGA ACC AT CAG CAC ATT CCT GAT GCC ACC T USP9X GTG TCA GTT CGT CTT GCT CAG C GCT GTA ACG ACC CAC ATC CTG A GAPDH GTC TCC TCT GAC TTC AAC AGC G ACC ACC CTG TTG CTG TAG CCA A 18S AGT CCC TGC CCT TTG TAC ACA GAT CCG AGG GCC TCA CTA AAC Supplementary Table 3: Primer sequences for real-time PCR analysis of mrna levels. 12
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27 Supplementary Figure 9: Western blot and capillary electrophoresis data. Uncropped western blots and capillary electrophoresis data from both the main and supplementary figures. 26
Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and
Figure S1. Analysis of genomic and sequences of the targeted regions in and indicated mutant KI cells, with WT and corresponding mutant sequences underlined. (A) cells; (B) K21E-KI cells; (C) D33A-KI cells;
a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53
1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1. Induction of p53 LOH by MADM. a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53 mouse revealed increased p53 KO/KO (green,
Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.
Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons
c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative
Supplemental Data. Shin et al. Plant Cell. (2012) /tpc YFP N
MYC YFP N PIF5 YFP C N-TIC TIC Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 Supplemental Figure. TIC interacts with MYC in the nucleus. Bimolecular fluorescence complementation assay using
Supplementary Figure 1 a
Supplementary Figure a Normalized expression/tbp (A.U.).6... Trip-br transcripts Trans Trans Trans b..5. Trip-br Ctrl LPS Normalized expression/tbp (A.U.) c Trip-br transcripts. adipocytes.... Trans Trans
Supplementary Materials
Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi
Supplementary Document
Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References 1. Supplementary Table legends Suppl.
Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at
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Supplementary Figures
Supplementary Figures Supplementary Figure 1. H3F3B expression in lung cancer. a. Comparison of H3F3B expression in relapsed and non-relapsed lung cancer patients. b. Prognosis of two groups of lung cancer
Supplementary Appendix
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Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most
Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most differentially expressed between human synovial fibroblasts
Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC-3 5 - GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA
Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
Supplementary Figure 1 U1 inhibition causes a shift of RNA-seq reads from exons to introns. (a) Evidence for the high purity of 4-shU-labeled RNAs used for RNA-seq. HeLa cells transfected with control
SUPPLEMENTARY DATA. Supplementary Table 1. Primer sequences for qrt-pcr
Supplementary Table 1. Primer sequences for qrt-pcr Gene PRDM16 UCP1 PGC1α Dio2 Elovl3 Cidea Cox8b PPARγ AP2 mttfam CyCs Nampt NRF1 16s-rRNA Hexokinase 2, intron 9 β-actin Primer Sequences 5'-CCA CCA GCG
CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'
Table S1. The primer sets used for real-time RT-PCR analysis. Gene Forward Reverse VEGF PDGFB TGF-β MCP-1 5'-GTT GCA GCA TGA ATC TGA GG-3' 5'-GGA GAC TCT TCG AGG AGC ACT T-3' 5'-GAA TCA GGC ATC GAG AGA
Supplementary Table 2. Conserved regulatory elements in the promoters of CD36.
Supplementary Table 1. RT-qPCR primers for CD3, PPARg and CEBP. Assay Forward Primer Reverse Primer 1A CAT TTG TGG CCT TGT GCT CTT TGA TGA GTC ACA GAA AGA ATC AAT TC 1B AGG AAA TGA ACT GAT GAG TCA CAG
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