Supporting Information

Stahl et al. 10.1073/pnas.1512863112 SI Text S1: ZFN-Mediated Inactivation of DPH Genes in MCF7 antibodies did not generate signals in Western blots of wild-type Cells MCF7, but revealed good signals without background in diph- The ZFN recognition sequences are derived from NM_001383.3 thamide-deficient MCF7. This antibody (MGb) can therefore (DPH1-wt), NC_000001.11 (DPH2-wt), NC_000003.12 (DPH3-wt), specifically detect eEF2 without diphthamide modification. NM_181706.4 (DPH4-wt), BC053857.1 (DPH5-wt), NM_080650.3 SI Text S3: eEF2 Diphthamide Modification and ADP (DPH6-wt), and NC_000009.12 (DPH7-wt). ZFN were obtained Ribosylation in MCF7 Cells from Sigma and transfected into MCF-7 cells in six-well dishes. Mutated clones were either obtained by isolating survivor clones Cell extracts for analyses of eEF2 modifications were prepared by following exposure to lethal doses of Pseudomonas exotoxin A, or lysing cells (4 × 105 cells in six-well dishes) in RIPA buffer. For by PCR-based HRM analyses (Fig. S1A). MS analyses, 100 μg of proteins were reduced with DTE and For toxin selection, cells were treated 48 h after transfection alkylated using iodoacetamide and further digested with trypsin. with 100 nM PE and further propagated to generate toxin- Resulting peptides were desalted using solid-phase extraction resistant colonies. These were isolated and recloned from single and ∼1 μg of the digest was analyzed by nanoLC-electrospray cells. For the genetic screen, gene-specific PCR fragments were ionization-MS using Parallel Reaction Monitoring on a Thermo generated and subjected to HRM, which marks mutation con- Q-Exactive Plus mass spectrometer (Thermo Electron). The taining clones by biphasic melting curves (Fig. S1A). most abundant precursor ions of the tryptic peptides F702-R716 The sequences of the alleles in clones generated by both containing the native or modified His715 at mass-over-charge procedures were determined by characterizing plasmids con- 437.2234 (native peptide), 462.4854 (ACP-derivative), and 472.7511 taining PCR-fragments derived from genomic DNA encom- (diphthamide-derivative) were selected and fragmented. The EF2 B passing the ZFN-target regions. Fig. S1 shows the ZFN binding peptides T287-K298 (mass-over-charge 747.9047), V415-K425 (mass- sites on DPH genes with positions indicated in the WT sequence. over-charge 554.3241), G -K (mass-over-charge 532.2928), and “ ” 667 675 ZFN cleavage sites are underlined. Uni-allelic knockouts ( i and A -R (mass-over-charge 900.4518) were coanalyzed to nor- “ ” 785 800 ii in the entries in Fig. S1B) contain one wild-type sequence and malize EF2 amounts across preparations. All PRM chromatograms one with out-of-frame insertions or deletions. Complete knock- were processed using Skyline 2.6 (46). “ ” “ ” outs ( iii and iv in the entries in Fig. S1B) contain two mutated For antibody-mediated determination of absence of eEF2 “ ” “ ” sequences ( a and b in the entries in Fig. S1B) with out-of- modification, an in-house generated (Roche Diagnostics) (SI Text frame insertions or deletions. Table S1 summarizes the individual S2) rabbit monoclonal antibody was applied. Western blot clones that were obtained. Further characterizations of parent and analyses of cell extracts with this antibody enables the determi- knockout cells are described in SI Text S2 (growth analyses nation of relative amounts of eEF2 without diphthamide. To and chromosome composition) and SI Text S3 (transcriptome detect actin and total human eEF2, monoclonal anti–β-Actin and TNF-pathway analyses by mRNAseq and FACS). (AC-74, Sigma) and eEF2 (H-118, Santa Cruz) were applied. SI Text S2: A Monoclonal Rabbit Antibody That Specifically Secondary antibodies coupled to horseradish peroxidase were Detects Unmodified eEF2 purchased from Dako. For detection of ADP ribosylation of A rabbit immunization and subsequent B-cell-cloning procedure eEF2, cell extracts were incubated with PE as toxin/enzyme and was applied to generate antibodies that specifically bind eEF2 biotinylated NAD as substrate, followed by detection of Bio- without diphthamide: Rabbits were immunized with a peptide ADPR-eEF2 in Western blots (32). spanning amino acids 708–724 of human eEF2 (TLHADAIH- SI Text S4: Characterization of DPH-Deficient Cells RGGGQIIPT, without diphthamide modification) coupled to Growth of Parent and Mutated MCF7. KLH. After 10 rounds of immunization with adjuvant, B cells Growth of parent and mu- expressing peptide-binding antibodies were isolated by FACS tated MCF-7 was assessed by seeding 10,000 cells in flat-bottom (using fluorescence-labeled eEF2 peptide), and subsequently 96-well plates and incubation at 37 °C in humidified 5% CO2. converted to recombinant antibody clones via B-cell cloning and Twenty-four hours after seeding, cells were exposed to toxins. To PCR-mediated V-region extraction (47). ELISA and Biacore determine cell growth, a cell proliferation assay was performed ’ analyses with eEF2 peptide as antigen and scrambled peptides as according to the manufacturer s specifications (CellTiter 96 controls were used to select antibody candidates. Western blot Aqueous One Solution Cell Proliferation Assay, Promega). analyses (peptide and purified eEF2 and eEF2 detection in Proliferation (DNA replication) was addressed by BrdU incor- MCF7 cell extracts) were performed to evaluate eEF2 specificity poration assays (Roche Diagnostics, Mannheim FRG) 72 h after and to identify those that selectively recognize eEF2 without toxin exposure. Fig. S4A shows that heterozygous gene inactiva- diphthamide. Fig. S2 shows Western blots of eight different tion has no effect on cell growth and complete inactivation of antibody candidates on extracts from parent MCF7 cells (contain DPH1, DPH2, and DPH4 does not affect growth or affects growth only diphthamide-modified eEF2), and MCF7 DPH1ko-ko cells, only slightly. Complete inactivation of DPH5 is tolerated by which contain only eEF2 without diphthamide modification as MCF7 cells, however, accompanied by a reduction of growth rate. determined by MS analyses (Fig. 1 and Fig. S3): of the eight analyzed antibodies, five detected eEF2 with low background in Sensitivity of MCF7 Derivatives to CHX and Saporin. Sensitivity of extracts of both cell lines. These bind eEF2 independent of MCF7 derivatives to CHX and saporin was adressed to analyze if presence or absence of the diphthamide and can be applied as inactivation of DPH genes (or loss of diphthamide) influences eEF2 control reagents. Two antibodies (MGa and MGd) gen- susceptibility toward protein synthesis inhibitors in a nonspecific erate stronger eEF2 signals with extracts of DPH-inactivated manner. Fig. S4B shows that loss of diphthamide has no effect on cells than wild-type cells. These antibodies bind better to non- cellular susceptibility toward non-ADP ribosylating protein syn- diphthamide eEF2 than to diphthamide eEF2 but are not spe- thesis inhibitors, such as CHX (Fig. S4B, Left) or saporin (Fig. cific for diphthamide-deficient eEF2. Finally, one of the eight S4B, Right).

Stahl et al. www.pnas.org/cgi/content/short/1512863112 1of6 Chromosome Composition. Chromosome composition was ana- following the manufacturer’s instructions. Sequencing libraries lyzed by subjecting metaphase spreads to FISH analyses with were quantified and quality controlled on Bioanalyzer using BACs that contained human DPH genes: BAC RP11-694J09 for High Sensitivity chips (Agilent Technologies) and on Qubit using huDPH1, BAC RP11-662O03 for huDPH2, BAC RP11-608O8 dsDNA HS Assay Kit (Life Technologies). All libraries were for huDPH3, BAC RP11-627B05 for huDPH4, BAC RP11- pooled and sequenced in four lanes on a HiSeq2500 sequencer 376O20 for huDPH5, BAC RP11-879B24 for huDPH6, and BAC (Illumina) for 2 × 50 cycles using version 3 cluster generation kits RP11-734A17 for huDPH7. Fig. S4C shows that the chromosome and version 3 sequencing reagents. As a sequencing control, 10% composition and DPH gene copy number remains constant, when of PhiX control library (Illumina) was spiked into each lane. comparing the parent and mutated MCF7 cells. Thus, the ZFN Reads were aligned to the human genome (hg19) using GSNAP mutagenesis procedure addressed selectively the target genes and (bioinformatics.oxfordjournals.org/content/26/7/873.abstract), with did not influence overall chromosome content, distribution, or splice junctions and exons being defined based on Ensembl v73. DPH gene copy number. Suitable metaphase spreads for complete Expression was then profiled using in-house tools and reads per DPH5 knockout cells could not be obtained, possibly reflecting the “ ” kilobase and million mapped read values were computed as pro- reduced growth phenotype of complete DPH5 inactivation. posed by Mortazavi et al. (48). We achieved more than 20M SI Text S5: Preactivation of Death Receptor Pathways in aligned reads per sample of which more than 50% aligned to Diphthamide-Deficient MCF7 exons. Differential expression was computed using DESeq (www. genomebiology.com/2010/11/10/r106) and differentially expressed To analyze if TNF hypersensitivity upon DPH gene inactivation is j j > < caused by increased levels of TNF-receptors on their surfaces, genes ( log2 ratio 1 and corrected p-Value 0.01) were ana- parent MCF7 and derivatives with complete DPH2 or DPH5 lyzed using Ingenuity Pathway Analysis. Expression patterns that knockouts were subjected to FACS analyses using TNFR-specific are associated with diphthamide deficiency and cause TNF hy- antibodies. Fig. S5A shows that the TNFR-specific cell surface persensitivity should be similar in DPH5ko and DPH2ko, yet be signals are indistinguishable between parent and mutated MCF7 different in parent MCF7. Therefore, we identified genes that > + < − < cells. Thus, TNF hypersensitivity is not explained by altered become differently expressed ( log 1, log 1, P 0.01) between TNFR levels. parent and DPH2ko cells, and between parent and DPH5ko To analyze if DPH gene inactivation is accompanied by al- cells. A comparison of both gene sets revealed an overlap of terations in intracellular gene-expression patterns, parent MCF7 gene expression/pathway patterns that become induced upon cells and derivatives with complete DPH2 or DPH5 knockouts inactivation of DPH2 and DPH5 (Fig. 3, Fig. S5B, and Table S2). were subjected to mRNA sequencing. Total RNA was extracted Of 50 genes with highest induction levels upon inactivation of and purified using the High Pure RNA Isolation Kit (Roche) DPH2 or DPH5, 11 were identical (i.e., highly induced upon in- according to the manufacturer’s instructions. For all samples, activation of diphthamide synthesis, irrespective of which gene has high-quality RNA was obtained (RNA integrity number > 9.5). been compromised). The most prominent markers for DPH2 and RNAseq libraries were prepared from 250 ng of total RNA using DPH5 inactivation in MCF7 are strongly induced expression of the TruSeq Stranded Total RNA preparation Kit (Illumina) TGFβ and TNFSF15.

Stahl et al. www.pnas.org/cgi/content/short/1512863112 2of6 Fig. S1. Generation of MCF7 derivatives. (A) ZFN transfected cells are either exposed to PE followed by isolation and characterization of surviving cells (Left branch), or individually cloned and subjected to PCR-based genetic analyses without toxin exposure (Right branch and example for HRM analyses below). (B) ZFN target sequences and allele sequences of mutated MCF7 clones.

Stahl et al. www.pnas.org/cgi/content/short/1512863112 3of6 th tal. et Stahl www.pnas.org/cgi/content/short/1512863112 i.S2. Fig. etr ltietfcto fatbde htseiial eeteF ihu diphthamide. without eEF2 detect specifically that antibodies of identification blot Western i.S3. Fig. Sbsddtriaino E2H1 modifications. H715 eEf2 of determination MS-based sample MGh MGd MGb MGe MGa MGg MGc MGf recRabbit MCF-7 wt mAb

DPH1ko

MCF-7 wt Acn An-

DPH1ko 4of6 MCF- 7 w t MCF- 7 w t DPH4 w t-ko A DPH1 ko-ko DPH1 w t-ko DPH5 w t-ko DPH2 ko-ko ko-ko DPH2 w t-ko DPH6 w t-ko wt-ko DPH4 ko-ko DPH3 w t-ko DPH7 w t-ko DPH5 ko-ko 3,4 2,2 3 2 2,6 1,8 (490nm) (490nm) 1,6 2,2

1,4 1,8

Absobance 1,4

Absobance 1,2

1 1 0 20 40 60 0 20 40 60 Hours Hours

B 1 MCF-7 wt 1 MCF-7 wt DPH1 ko-ko DPH1 ko-ko DPH2 ko-ko DPH2 ko-ko 0,8 DPH4 ko-ko 0,8 DPH4 ko-ko DPH5 ko-ko DPH5 ko-ko 0,6 0,6

0,4 0,4

cell viability 0,2 ko-ko0,2 ko-ko

0 0 10 CHX 100 1000 10000 [nM] 10 Saporin 100 1000 10000 [nM]

MCF-7 wt 1,2 MCF-7 wt DPH1 wt-ko DPH1 wt-ko 1,2 DPH2 wt-ko 1 DPH2 wt-ko DPH3 wt-ko DPH3 wt-ko 1 DPH4 wt-ko 0,8 DPH4 wt-ko DPH5 wt-ko DPH5 wt-ko 0,8 DPH6 wt-ko 0,6 DPH6 wt-ko 0,6 DPH7 wt-ko DPH7 wt-ko 0,4 0,4

cell viability wt-ko wt-ko 0,2 0,2 0 0 10 CHX 100 1000 10000 [nM] 10 Saporin 100 1000 10000 [nM]

C DPH1 DPH2 DPH3DPH4 DPH5 DPH6 DPH7

wt

wt-ko

ko-ko

Fig. S4. (A) Growth, (B) susceptibility toward saporin and CHX, and (C) chromosome composition of MCF7 derivatives. (C) Images were captured with a Zeiss Plan-Neofluar microscope at 100× magnification.

Stahl et al. www.pnas.org/cgi/content/short/1512863112 5of6 Fig. S5. (A) Analyses of TNFR expression by FACS with TNFR specific antibodies. (B) Transcriptome (mRNAseq) comparisons of MCF7.

Table S1. Summary of the individual clones that were obtained No. of clones toxin No. of clones Gene No. of transf. cells (t or g*) selection genetic screen

DPH1 4 × 105 (t) 3 (KO-KO) 4 (WT-KO) 1,6 × 106 (g) 2 (KO-KO) DPH2 4 × 105 (t) 3 (KO-KO) 4 (WT-KO) 2 × 106 (g) 0 (KO-KO) DPH3 3.6 × 106 (t) 0 5 (WT-KO) 3.6 × 106 (g) 0 (KO-KO) DPH4 4 × 105 (t) 3 (KO-KO) 2 (WT-KO) 1.2 × 106 (g) 1 (KO-KO) DPH5 4 × 105 (t) 3 (KO-KO) 3 (WT-KO) 2.4 × 106 (g) 1 (KO-KO) DPH6 3.6 × 106 (t) 0 4 (WT-KO) 1.6 × 106 (g) 0 (KO-KO) DPH7 3.6 × 106 (t) 0 3 (WT-KO) 3.6 × 106 (g) 0 (KO-KO)

“t” or “g” indicates number of transfected cells subjected to toxin selection (t) and genetic screen (g). “No. of clones” lists the number of completely (KO-KO) or heterozygous (WT-KO) mutated individual clones (i.e., with different mutations) that were identified by sequence analyses of clones obtained by toxin selection or via HRM curve shapes in genetic screens.

Table S2. Top-inductions as a consequence of DPH2 and DPH5 DPH2ko DPH5ko

Gene Rank Value Rank Value

TGFB2 4 −4.9 2 −9.5 TNFSF15 6 −4.8 1 −10.3 CLDN1 9 −4.5 11 −7.3 Sulf1 10 −4.4 18 −6.5 Slc2a12 17 −4.1 28 −5.9 MCF2L2 21 −4.0 37 −5.7 Krt87p (pseudogene) 22 −4.0 21 −6.4 PDE10A 23 −3.9 29 −5.9 S100A9 27 −3.8 48 −5.5 GABARAPL1 40 −3.5 46 −5.5 ALDH1A3 50 −3.6 20 −6.4

Top-inductions as a consequence of DPH2 as well as DPH5 inactivation were defined by calling transcripts that

are among the 50 genes (rank) with highest induction levels (log2 value) compared with parent MCF7 in DPH2ko cells, as well as in DPH5ko cells. The most prominent markers for DPH2 and DPH5 inactivation in MCF7 are induction of TGFβ and TNFSF15.

Stahl et al. www.pnas.org/cgi/content/short/1512863112 6of6