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1 DOI: /ncb1816 A Gal4 Gal4 B Kinase domain C (1502) N ( ) Interaction with SIAH1 C SIAH1 SIAH1 C (182) SIAH1 N (82282) RING domain Interaction with Input GST GSTSIAH1 1a a N C Input GST GSTSIAH1 GSTSIAH1 C GSTSIAH1 N 1a C N a Coomassie a GSTSIAH1 D a GST Coomassie E a a GSTSIAH1 GSTSIAH1 N GSTSIAH1 C GST IP: IgG IP: IgG HASIAH1 RM Flag HASIAH2 RM Flag a SIAH1 a SIAH2 1a 1a INPUT: HASIAH1 RM Flag a SIAH1 INPUT: HASIAH2 RM Flag a SIAH2 1a 1a Figure S1 (A) Yeast cells expressing a Siah1Gal4 activation domain hybrid protein were transformed with vectors directing the expression of either the DNAbinding domain of Gal4 alone or a Gal4 fusion protein lacking the speckle retention sequence. The yeast cells were tested for the activity of a Gal4dependent lacz reporter gene as shown. (B) In vitro translated and [ 35 S] labeled and truncated versions thereof were tested for interaction with bacterially produced GST and GSTSiah1 proteins as shown. The upper part shows schematic representations of and its mutants, the lower part displays an autoradiogram and the Coomassiestained input material. (C) Purified GST, GSTSiah1 and its derivatives were incubated with that was [ 35 S] labeled by in vitro translation. An autoradiogram and input control (lower) and a schematic representation of Siah (upper) are shown. (D) Human 293T cells were transfected to express Flagtagged along with an HAtagged Siah1 RM as shown. A fraction of the cell lysates was tested for the correct expression of the transfected proteins by Western blotting (WB), as shown in the lower part. The remaining extracts were used for immunoprecipitation (IP) with αflag antibodies or isotypematched control antibodies. After elution of bound proteins in 1 x SDS sample buffer, coprecipitated Siah1 was visualized by immunoblotting as shown. (E) The experiment was performed as in (D) with the exception that an expression vector encoding Siah2 RM was used. (F) Human 293T cells were transfected to express Flagtagged along with an HAtagged Siah2 as shown. The next day, cells were incubated for 10 h with MG132, followed by cell lysis and testing for the correct expression of the transfected proteins by immunoblotting, as shown in the lower part. The remaining extracts were used for immunoprecipitation with αha antibodies or isotypematched control antibodies. After elution of bound proteins in 1 x SDS sample buffer, coprecipitated was revealed by immunoblotting as shown. (G) Cells transfected to express Flag and increasing amounts of Siah2 RM were grown in the presence or absence of MG132, followed by lysis of cells in 1 x SDS lysis buffer and Western blot analysis as shown. (H) The experiment was performed as in (G) with the exception that Siah2 wildtype was expressed. (I) Cells were transfected with an expression vector encoding Flag and increasing amounts of Siah2 as shown. 36 h later, cells were harvested and one half was used to prepare protein lysates that were further subjected to Western blot analysis using the indicated antibodies. RNA was isolated from the remaining half of cells and relative mrna levels of and the control were determined by RTPCR with specific primers. An ethidium bromidestained agarose gel is displayed. Uncropped images of the key scans in B, C, D, E, F, G and I are shown in Supplementary Information, Fig. S6. 1

2 F G IP: Flag HASIAH2 a IgG Flag HASIAH2 RM MG132 1a Marco: please show also more of the space INPUT: Flag HASIAH2 MG132 a a a SIAH2 a SIAH2 H I Flag HASIAH2 MG132 Flag HASIAH2 a 1a Ubi* 1a a SIAH2 a a Ubi*SIAH2 SIAH2 RTPCR: a Figure S1 continued 2

3 A Flag Flag 1967 Flag NLS HASIAH2 a a a SIAH2 B Flag DAPI Merge Flag 1967 DAPI Merge Flag NLS DAPI Merge Figure S2 (A) Flagtagged or its derivatives were expressed alone or together with Siah2 as shown. After 36 h, cells were lyzed and tested for degradation by immunoblotting. (B) Cells were transfected with the indicated plasmids and analyzed for localisation by indirect immune fluorescence. Nuclear DNA was stained with DAPI as shown. Scale bar = 10 µm. (C,D) The HAtagged Siah2 RM protein was coexpressed with or its various mutant forms as indicated. Cell lysates were subjected to immunoprecipitation with anti HA antibodies or isotypematched control antibodies. Bound proteins were eluted and further analyzed by immunoblotting as shown. An aliquot of the input material was also tested for correct protein expression. (E) Cells were transfected to express increasing amounts of Siah2 along with various Flagtagged mutants which are schematically shown on the left. stability was determined by immunoblotting using anti Flag antibodies. Adequate Siah2 expression and equal protein loading was ensured in every single experiment (data not shown). (F) or the degradation resistant form of the kinase were coexpressed with Siah2 and Histagged ubiquitin at the indicated combinations. The next day, MG132 was added and protein expression and modification were revealed by Western blot analysis. Ubiquitination was shown after purification of ubiquitinated proteins on Ni NTA columns. (G) or a variant mutated in all lysines between amino acids 796 and 994 ( K11/R) were expressed alone or together with limited amounts of Siah2. Degradation and ubiquitination was analyzed by Western blotting. Uncropped images of the key scans in A, C, D, E, F, and G are shown in Supplementary Information, Fig. S6. 3

4 C Flag Flag Flag Flag 1967 HASIAH2 RM IP: IgG a a a INPUT: a a a a SIAH2 D Flag Flag 1729 Flag 1786 Flag Flag 1502 HASIAH2 RM IP: IgG a a a a INPUT: a a a a a SIAH2 Figure S2 continued 4

5 E a a a a Kinase domain 1191 Flag HASIAH2 a a a a SIAH2 Degradation by SIAH2 F Flag HASIAH2 HisUbi MG132 Flag HASIAH2 HisUbi MG132 a 1a Ubi* a 1a Ubi* a Ubi*SIAH2 a Ubi*SIAH2 a SIAH2 a SIAH2 a a a 1a Ubi* NiNTA purification a 1a Ubi* NiNTA purification Ubi 1a Ubiquitin Ubi 1a Ubiquitin a a Figure S2 continued 5

6 G Flag Flag K11/R HASIAH2 HisUbi MG132 a 1a Ubi* a a SIAH2 Figure S2 continued 6

7 A Flag Flag K1184A Flag DD916/977AA HASIAH2 HisUbi MG132 a Ubi* 1a a a SIAH2 B C Flag HASIAH2 ADR zvad FlagSIAH2 HA HA K221A MG132 1a a P SIAH2 SIAH2 a SIAH2 1a K221A a a phosphatase D FlagSIAH2 HA MG132 S5A S10A T26A S28A S68A T113A T119A S121A S279A a P SIAH2 SIAH2 1a a Figure S3 (A) Cells were transfected to express Flag, K1184A or the caspase resistant DD/AA mutant along with Siah2 or Histagged ubiquitin as shown. Cells were further cultivated in the absence or presence of MG132 and analyzed for the stability and ubiquitination of by Western blotting. (B) Flag was expressed either alone or along with Siah2. Cells were further incubated with 20 µm of zvadfmk and analyzed for protein expression as shown. The functionality of this broad spectrum caspase inhibitor was ensured in a control experiment, as the published caspasedependent and adriamycintriggered cleavage of (lane 2) was completely prevented by zvadfmk (lane 3). (C) Cells were transfected to express the indicated combinations of Siah2 and proteins in the presence of MG132. The sample analyzed for the Siah2 phosphorylation was lyzed in phosphatase inhibitorfree buffer containing λ phosphatase as shown. The electrophoretic mobility of Siah2 was determined by immunoblotting. (D) The indicated Siah2 point mutants were coexpressed with in the presence of MG132 as shown and analyzed for their electrophoretic mobility by immunoblotting. Uncropped images of the key scans in A, B, C, and D are shown in Supplementary Information, Fig. S6. 7

8 A B Hypoxia (h) 6 12 Flag Hypoxia psupersiah2 1a a RTPCR: SIAH2 1a a a SIAH2 C D Flag Hypoxia Flag HASIAH2 HASIAH1 Hypoxia SIAH2 a a SIAH2 a a SIAH2 SIAH1 a a SIAH1a / cells SIAH1a/2 / cells Figure S4 (A) 293 cells were grown under normoxic or hypoxic conditions (1% O 2 ). One aliquot of the cells was lyzed and tested for protein expression (upper), another aliquot was used for mrna isolation, followed by the determination of mrna levels for and the control by RT PCR (lower). (B) 293 cells were transfected with psupersiah2, a vector directing the synthesis of Siah2 specific shrna or with the empty vector as a control. After 4 days, cells were cultivated under normoxic or hypoxic conditions and analyzed for stability and Siah2 abundance as shown. (C) MEFs expressing and deficient either for Siah1a or Siah1a/Siah2 were incubated for 12 h under normoxic or hypoxic conditions, followed by the analysis of abundance as shown. (D) Siah1a/Siah2deficient cells were stably retransfected to express low physiological levels of Siah1 or Siah2 as shown. Cell pools were then transfected with a expression vector, followed by 12 h incubation under normoxic or hypoxic conditions and analysis of protein expression by immunoblotting. Uncropped images of the key scans in A, B, C, and D are shown in Supplementary Information, Fig. S6. 8

9 A HA FlagSIAH2 FlagSIAH2 TSS/AAA 1a a a P SIAH2 SIAH2 B IP: IgG HA FlagSIAH2 Hypoxia MG132 INPUT: HA FlagSIAH2 Hypoxia MG132 1a 1a a SIAH2 a P SIAH2 SIAH2 C IP: IgG HA FlagSIAH1 Hypoxia MG132 INPUT: HA FlagSIAH1 Hypoxia MG132 1a 1a a SIAH1 a SIAH1 D FlagSIAH2 FlagSIAH2 S28A HA MG132 E psuper P SIAH2 P SIAH2 a 1a a P SIAH2 SIAH2 SIAH2 P a a P SIAH2 103 kda Figure S5 (A) Cells were transfected with expression vectors encoding HA and Flag tagged versions of Siah2 or Siah2 TSS/AAA as shown. The next days cells were lyzed and the stability of was revealed by immunoblotting. (B) Cells transfected to express epitope tagged versions of and Siah2 were incubated for twelve hours in the presence of MG123 under normoxic or hypoxic conditions. One aliquot was analyzed for adequate protein expression (right), another aliquot was tested for mutual interaction of both proteins by coimmunoprecipitation (left). (C) Effect of hypoxia on /Siah1 interaction. Cells were transiently transfected to express tagged versions of and Siah1, followed by the induction of hypoxia in the presence of MG132 as shown. One aliquot was analyzed for adequate protein expression (right), another aliquot was tested for mutual interaction of both proteins by coimmunoprecipitation (left). Following immunoprecipitation of Siah1, the amount of associated was determined by Western blotting. (D) Characterisation of the Siah2 phosphospecific antibody. Flagtagged Siah2 or the point mutant where ser 28 was changed to ala were expressed either alone or along with in 293T cells. Lysates were tested for adequate protein expression and Siah2 ser 28 phosphorylation with the indicated antibodies. (E) Increasing amounts of psuper, a vector producing a specific shrna, were introduced into 293T cells. After three days, equal amounts of protein contained in lysates were analyzed for protein expression and Siah2 phosphorylation. (F) Cells were transfected with the indicated expression vectors. After further incubation under normoxic or hypoxic conditions, cells were harvested. One aliquot of the cells was checked for protein expression by Western blotting (lower), while another aliquot was analyzed for cyp1b1 gene expression by real time PCR (upper). (G) Effects of Siah phosphorylation on gene expression. 293TRex cells expressing almost undetectable levels of endogenous Siah2 were transfected with expression vectors encoding, Siah2 or Siah2 S28A. Cells were checked for protein expression by Western blotting (lower) and for cyp1b1 gene expression by real time PCR (upper). All gene expression experiments in this figure were performed in triplicates, error bars show standard deviations (p < 0.05 were observed in all the samples). Uncropped images of the key scans in A, B, C, D, E, F and G are shown in Supplementary Information, Fig. S6. 9

10 F Fold Induction CYP1B1 Hypoxia HASIAH2 Flag Flag a a G Fold Induction 8 CYP1B HA FlagSIAH2 FlagSIAH2 TSS/AAA 1a a a P SIAH2 SIAH2 Figure S5 continued 10

11 1 Figure 1A 1 Figure 1B 1 1 Figure 1C 1 1 IB: siah1 IB: siah2 IB: HA IB: IB: Ubi 1 IB: 1 Figure 2A 1 1 IB: HA Figure 2B Figure 2C 1 Figure 2E 1 1 Figure 3A IB: Kinase assay assay 32 P Figure 3B Figure 2D Coomassie staining i i IB: 1 Figure S6 Full scan of the key western blots from the figures indicated. 11

12 Figure 3C Figure 3D Figure 4B Figure 4C IB: Figure 4A 1 IP: siah2 IB: IB:pSiah IP: Flag IB:HA IB:siah2 Figure 4d IB: psiah2 Figure 4E IP: HA Figure 5C 1 IB: Figure 5D IB:siah2 Figure 5A 1 1 Figure 5E IB: IB: pp53 ser 46 Figure S6 continued 12

13 Figure S1B Figure S1C Figure S1E 1 1 Autoradiogram [ 35 S] Autoradiogram [ 35 S] IP: Flag IB:HA Figure S1D 1 1 Figure S1F Figure S1G 1 IP: Flag IB:HA IP: HA Figure S1I Figure S2A Figure S2C Figure S2D Figure S2E IP: HA IP: HA IB: Figure S6 continued 13

14 Figure S2F Figure S2G Figure S3A Figure S3B 1 1 IB:HA IB: Figure S3C 1 Figure S3D 1 Figure S4B 1 IB:siah2 Figure S4A Figure S4C Figure S4D Figure S6 continued 14

15 Figure S5A Figure S5B Figure S5C Figure S5D IB:HA 1 IP:Flag IB:HA Figure S5F 1 IP:Flag IB:HA IB:psiah2 IB: Figure S5E 1 IB: IB:psiah2 Figure S5G Figure S6 continued 15

16 Supplementary Experimental Procedures Yeast twohybrid analysis A mutant of human bearing two point mutations (KK2221/1184AA) and deficient for the speckle retention signal was fused to the Gal4 DNAbinding domain in the yeast expression vector pgbkt7. The yeast strain AH109 containing this bait vector was transformed with a human leukemia matchmaker cdna library (pact2), selected for growth on Ade/His/Leu/Trpdeficient medium and tested for lacz expression according to the manual provided by the manufacturer (Clontech). interacting proteins encoded by cdnas contained in the pact2 vector were isolated from yeast cells, retransformed into E.coli and analyzed by sequencing. DNA arrays The oligonucleotide microarrays were performed using the Human Genome U133 Plus 2.0 Array (Affymetrix) system. 8 µg purified RNA were tested for their quality on an Agilent Bioanalyzer and reverse transcribed by Superscript II reverse transcriptase (Life Technologies) using T7(dT) 24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cdna) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (crna). Fifteen micrograms of fragmented crna was hybridized to a Human Genome U133 Plus 2.0 Array for 16 h at 45 C with constant rotation at 60 rpm. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an GeneChip Scanner 3000 (Affymetrix). Data analysis was performed with the Affymetrix Microarray Suite or GCOS and with Genespring software version 6 (Silicon Genetics, San Carlos, CA). The data were normalised as follow: values below 0.01 were set to Each measurement was divided by the 50,0th percentile of all measurements in that sample. Each gene was divided by the median of its measurements in all samples. If the median of the raw values was below 10 then each measurement for that gene was divided by 10 if the numerator was above 10, otherwise the measurement was thrown out.

17 In vitro kinase assays Recombinant GST and GSTSiah proteins were expressed in E.coli and purified by standard protocols. Flag and Flag K221A were isolated from transfected cells by immunoprecipitation using antiflag monoclonal antibodies and assayed for in vitro kinase activity as described 8. Cell culture and transfections 293, 293T, 293TRex, U2OS, MCF7, wildtype MEFs and Siahdeficient MEFs were grown in DMEM containing 10% FCS and 1% (w/v) penicillin/streptomycin at 37 o C and 5% CO2. Cells were transiently transfected using Rotifect (Roth) reagents. MEFs were transfected using the Amaxa nucleofection system or by electroporation according to the instructions of the manufacturers. Hypoxia was induced by cultivation of cells in a New Brunswick Innova48 incubator at defined O 2 concentrations.