Supporting Information

Slater et al. 10.1073/pnas.1302257110 SI Materials and Methods duplex and 0.9 μL of Lipofectamine RNAiMAX (Invitrogen) in Flow Cytometry. Approximately 1 million J774A.1 cells were washed 50 μL of Opti-MEM-1 (minimum essential medium) reduced se- three times in FACS buffer (PBS pH 7.4 and 0.5% FBS), stained rum medium (Gibco) and were made up according to the man- with anti-anthrax toxin receptor 2 (ANTXR2) antibody or normal ufacturer’s instructions. After 24 h, transfection medium was goat IgG (Santa Cruz), washed three times with FACS buffer, replaced with fresh culture medium, and cells were incubated stained with secondary Alexa Fluor 488 donkey anti-goat IgG an- a further 24 h before challenge with LT. tibody, washed three times with FACS buffer, fixedwith4%para- formaldehyde in PBS for 20 min at room temperature, and washed RT-PCR. J774A.1 cells in 96-well plates were infected with lentivirus twice with FACS buffer. Fluorescence was measured by using the encoding chaperonin containing t-complex protein 1 (TCP-1) 6a-1 FACSCalibur flow cytometer (Beckton Dickinson Biosciences). (CCT6a-1), CCT6b-2, or nontargeting shRNA, and knockdown was Data analysis was performed using BD CellQuest Pro Software. allowed to proceed for 7 d. Cells were lysed, and total RNA was isolated by using the RNeasy 96 kit (Qiagen) following the manu- siRNA Knockdown of ANTXR2. Interfering RNA siGENOME was facturer’s instructions. cDNA was synthesized from total RNA by obtained from Dharmacon. The catalog number for the non- adding 9-mer random primers, oligo-dT (20 mer), dNTPs, and targeting siRNA is D-001206-14. The siRNA sequence for the Moloney murine leukemia virus reverse transcriptase (Roche) and ANTXR1 pool was as follows: GAGGGACAACUUUAAUGAA, incubating for 50 min at 37 °C. RT-PCR was performed on 0.4 μLof GCGUUGGCGUGAAGGAUUU, GAGCAGAUUUACUA- cDNA in 10-μL volume by using SYBR Green-based detection on UGAGA, and GAAAGAAGUUGGCAUGAAA. The siRNA an ABI 7200 HT RT-PCR System (Applied Biosystems). Relative sequence for the ANTXR2 pool was as follows: ANTXR2-a expression of CCT6a and CCT6b was calculated by using the com- UUGCUUUGACGGACGGUAA, ANTXR2-b GACUAAAG- parative CT (threshold cycle) method with GAPDH as the reference CUUGCAAACGA, ANTXR2-c CAUCUUAUGCAGAGAA- mRNA. The primers used in the RT-PCR were as follows: CCT6a, CGA, and ANTXR2-d GAGAAUUAAUAGAGCGUUA. A 5′-GGCACTACATCCAATGTCCTC-3′ and 5′-TCGATGAG- total of 1.3 × 104 J774A.1 cells were plated in white, optical 96- TGTTTCTCTGTCCAT-3′; CCT6b, 5′CAAAGACGGCAAT- well plates in 100 μL and incubated overnight. Medium was re- GTACTGCT-3′ and 5′-GCCACATCCAAGAGGATTTCTC- placed with 50 μL of fresh culture medium and 50 μLoftrans- 3′;andGAPDH,5′-TGAAGCAGGCATCTGAGGG-3′ and 5′- fection mix. Transfection mixes contained 2 pmol of siRNA CGAAGGTGGAAGAGTGGGAG-3′.

Fig. S1. Expression and knockdown of CCT6a and CCT6b in J774A.1 cells. J774A.1 cells were infected with lentivirus encoding CCT6a-1, CCT6b-2, or non- targeting shRNA, and knockdown was allowed to proceed for 7 d. Cells were lysed, RNA was isolated, cDNA was synthesized, and mRNA was quantified by RT- PCR. The graphs show the amount of CCT6a (A) and CCT6b (B) transcript remaining compared with nontargeting control.

Slater et al. www.pnas.org/cgi/content/short/1302257110 1of6 Fig. S2. Knockdown of CCT in J774A.1 cells causes a small reduction in cell surface levels of ANTXR2. (A and B) ANTXR2 is the major toxin receptor mediating lethality in J774A.1 cells. (A) J774A.1 cells in 96-well format were transfected with individual or pooled siRNA (Dharmacon) by using Lipofectamine RNAiMax (Invitrogen) for 24 h at 37 °C. Knockdown was allowed to proceed for a total of 48 h before challenge with 11 nM protective antigen (PA) and 0.25 nM lethal factor (LF) for 6 h at 37 °C. Cell viability was assessed by adding Cell Titer Glo (Promega) and measuring luminescence using a SpectraMax M5 plate reader. (B) J774A.1 cells in 384-well plates were incubated with DMEM and 1% FBS containing 38 μg/mL anti-ANTXR2 antibody or goat IgG for 1 h at 4 °C. Lethal toxin (LT) (11 nM PA, 0.5 nM LF) was added and incubated for 6 h at 37 °C, followed by addition of Cell Titer Glo (Promega) and measurement of luminescence on a SpectraMax M5 plate reader. (C–E) CCT knockdown in J774A.1 cells plated in six-well plates was allowed to proceed for 5 d. Cells were stained with an anti- ANTXR2 antibody or goat IgG (negative control), followed by a rabbit anti-goat antibody conjugated to Alex Fluor 488, and analyzed by flow cytometry on a FACSCalibur Flow Cytometer (BD Biosciences). (C) Raw data showing fluorescence detected in anti-ANTXR2-stained cells. (D) Positively stained cell pop- ulations (dotted line) were identified by subtracting the histogram from IgG-stained cells (dashed line) from the histogram for anti-ANTXR2–stained cells (solid line). Data were analyzed by using BD CellQuest Pro software. (E) The number of positively stained cells expressed as a percentage of the total number of cells, calculated from the histograms shown in D.

Table S1. RNAi screen: Details of the lentiviral shRNA libraries Library No. of genes

Mouse kinase 939 Mouse phosphatase 322 Mouse vesicle trafficking 383 Mouse chaperone 136

Slater et al. www.pnas.org/cgi/content/short/1302257110 2of6 Table S2. RNAi screen: Hits from a custom library of genes No. of active shRNAs

>2SD >1SD Highest mean GeneID Symbol Description from mean from mean robust z score

60533 Cd274 CD274 antigen 1 1 56 56404 Trip4 Thyroid hormone receptor interactor 4 1 1 30 12362 Casp1 Caspase 1 2 4 16 12176 Bnip3 BCL2/adenovirus E1B interacting protein 3 1 2 12 170770 Bbc3 BCL2 binding component 3 1 1 16 74343 Crtc2 CREB regulated transcription coactivator 2 1 2 6 15466 Hrh2 Histamine receptor H2 0 2 5

BCL2, B cell leukaemia/lymphoma 2; BH3, BCL2 binding component 3; CREB, cAMP response element binding protein.

Table S3. RNAi screen: Hits from the chaperone library No. of active shRNAs

>2SD >1SD Highest mean GeneID Symbol Description Notes from mean from mean robust z score

12469 Cct8 Chaperonin containing Tcp1, subunit 8 (theta) CCT chaperonin complex 3 3 158 15502 Dnaja1 DnaJ [heat-shock protein 40 (Hsp40)] homolog, DnaJ/Hsp40 homolog 1 1 95 subfamily A, member 1 69408 Dnajc17 DnaJ (Hsp40) homolog, subfamily C, member 17 DnaJ/Hsp40 homolog; RNA 22 90 binding domain 12462 Cct3 Chaperonin containing TCP1, subunit 3 (gamma) CCT chaperonin complex 3 3 87 12468 Cct7 Chaperonin containing Tcp1, subunit 7 (eta) CCT chaperonin complex 3 3 50 12466 Cct6a Chaperonin containing Tcp1, subunit 6a (zeta) CCT chaperonin complex 2 3 48 12464 Cct4 Chaperonin containing Tcp1, subunit 3 (delta) CCT chaperonin complex 3 3 45 12465 Cct5 Chaperonin containing Tcp1, subunit 5 (epsilon) CCT chaperonin complex 3 4 38 12461 Cct2 Chaperonin containing TCP1, subunit 2 (beta) CCT chaperonin complex 1 1 31 12467 Cct6b Chaperonin containing Tcp1, subunit 6b (zeta) CCT chaperonin complex 3 3 27 14828 Hspa5 (GRP78) Heat shock protein 5; 78 kDa glucose-regulated Lethal factor unfolding 1 1 27 protein

Slater et al. www.pnas.org/cgi/content/short/1302257110 3of6 Table S4. RNAi screen: Hits from the vesicle trafficking library No. of active shRNAs

>2SD >1SD Highest mean GeneID Symbol Description Notes from mean from mean robust z score

71732 Vps11 Vacuolar protein sorting May play a role in late endosome-lysosome fusion 3 4 97 11 (yeast) 100972 Rab28 RAB28, member RAS Unknown 3 3 52 oncogene family 69834 Rab43 RAB43, member RAS May have a role in ER to Golgi or endosome to Golgi 11 39 oncogene family trafficking 53330 Vamp4 Vesicle associated membrane v-SNARE localized to Golgi. Involved in endosome-Golgi 22 33 protein 4 transport. Implicated in homotypic fusion of early endosomes 76983 Scfd1 Sec1 family domain Involved in vesicular transport between the endoplasmic 23 31 containing 1 reticulum and the Golgi. 13429 Dnm1 Dynamin 1 Involved in clathrin-mediated endocytosis and other 11 31 vesicular trafficking processes. Neuron-specific 56447 Copz1 Coatomer protein complex, COPI has been implicated in translocation of LF from 11 28 subunit zeta 1 endosome to cytosol 106039 Gga1 Golgi associated, gamma ARF binding protein 1, a cargo/clathrin adaptor protein. 22 27 adaptin ear containing, Involved in trafficking from the TGN to the lysosome, ARF binding protein 1 and from endosomes to the TGN. 58244 Stx6 Syntaxin 6 t-SNARE localized to the TGN. Implicated in endosome 11 25 to TGN trafficking along with VAMP4. 66700 Vps24 Charged multivesicular Member of the ESCRTIII complex involved in MVB 11 22 body protein 3 formation + 11975 Atp6v0a1 ATPase, H transporting, Proton pump controlling endosome acidification 3 3 20 lysosomal V0 subunit a1 50797 Copb2 Coatomer protein complex, COPI has been implicated in translocation of LF from 33 18 subunit beta 2 endosome to cytosol 13430 Dnm2 Dynamin 2 Involved in clathrin-mediated endocytosis and other 11 18 vesicular trafficking processes. Ubiquitously expressed. 227700 Sh3glb2 Src homology (SH3)-domain Endophlin. Contains a BAR domain that can cause 11 18 growth factor receptor- membrane curvature, therefore likely involved in bound protein 2 (GRB2)- clathrin-dependent endocytosis like endophilin B2 + 12890 Cplx2 Complexin 2 Complexins are SNARE regulators that control Ca2 11 18 dependent exocytosis 20479 Vps4b Vacuolar protein sorting AAA+ ATPase involved in intracellular protein trafficking 11 17 4b (yeast) through the endosome to the lysosome. Is recruited by ESCRTIII to MVBs 19344 Rab5b RAB5B, member RAS Rab5 GTPases localize to early endosomes and regulate 22 17 oncogene family fusion of clathrin-coated vesicles to early endosomes and fusion between early endosomes. 70747 Tspan2 Tetraspanin 2 Transmembrane protein shown to bind integrin. 22 15 Tetraspanins show lipid raft localization properties. 69908 Rab3b RAB3B, member RAS Required for Ca2+-regulated exocytosis 3 3 15 oncogene family 12391 Cav3 Caveolin 3 Caveolins are involved in vesicular transport, cholesterol 22 14 homeostasis, signal transduction, and tumor suppression. 19345 Rab5c RAB5C, member RAS See Rab5b 3 3 13 oncogene family 20981 Syt3 Synaptotagmin III Synaptotagmin 1 is involved in Ca2+ induced synaptic 22 13 vesicle fusion. Synaptotagmin 3 is localized to MVBs.

ARF, ADP-ribosylation factor; RAS, rat sarcoma; Sec1, secretory blood group 1; Src, Rous-sarcoma oncogene.

Slater et al. www.pnas.org/cgi/content/short/1302257110 4of6 Table S5. RNAi screen: Hits from the phosphatase library No. of active shRNAs

>2SD >1SD Highest mean GeneID Symbol Description from mean from mean robust z score

18186 Nrp1 Neuropilin1 1 2 234 268783 Mtmr12 Myotubularin related protein 12 1 1 78 19270 Ptprg Protein tyrosine phosphatase, receptor type, G 1 1 73 17288 Mep1b Meprin 1 beta 1 1 49 93760 Arid1a AT rich interactive domain 1A (SWI-like) 1 2 46 20975 Synj2 Synaptojanin 2 1 1 35 19257 Ptpn3 Protein tyrosine phosphatase, nonreceptor type 3 2 3 34 19043 Ppm1b Protein phosphatase 1B, magnesium dependent, 11 33 beta isoform 19252 Dusp1 Dual specificity phosphatase 1 1 2 22 19281 Ptprt Protein tyrosine phosphatase, receptor type, T 2 2 20 237178 Ppef1 Protein phosphatase with EF hand calcium-binding 23 14 domain 1 320634 Ocrl Oculocerebrorenal syndrome of Lowe 2 3 28

Table S6. RNAi screen: Hits from the kinase library No. of active shRNAs

>2SD >1SD Highest mean GeneID Symbol Description from mean from mean robust z score

13840 Epha6 Ephrin receptor A6 1 1 233 18793 Plaur Plasminogen activator, urokinase receptor 1 1 167 71340 Riok1 RIO kinase 1 1 1 152 217837 Itpk1 Inositol 1,3,4-triphosphate 5/6 kinase 3 3 134 76367 Trp53rk Transformation related protein 53 12108 regulating kinase 545204 Gm318 Predicted gene 318 1 1 98 56174 Nagk N-acetylglucosamine kinase 1 1 94 59126 Nek6 NIMA-related expressed kinase 6 1 1 72 109042 Prkcdbp Protein kinase C, delta binding protein 1 1 51 60596 Gucy1a3 Guanylate cyclase 1, soluble, alpha 3 1 2 38 22367 Vrk1 Vaccinia related kinase 1 2 2 28 78891 Scyl1 Preprotein translocase subunit secretion (sec)Y 22 22 (SCY1)-like 1 (S. cerevisiae) 54124 Cks1b CDC28 protein kinase 1b 1 2 20 214253 Etnk2 Ethanolamine kinase 1 2 17

CDC28, cell division cycle 28; NIMA, never in mitosis gene a; RIO, right open reading frame.

Slater et al. www.pnas.org/cgi/content/short/1302257110 5of6 Table S7. RNAi screen: shRNAs targeting CCT subunits that conferred resistance to LT shRNA Mean robust z score Target sequence TRC clone ID

Cct2-1 30.8 GCTCACAGTGAAGGCCATATA TRCN0000120469 Cct3-1 87.1 AGGAACACTGTGGACATCTTT TRCN0000120447 Cct3-2 59.2 GCAGCAAAGTCCATGATTGAA TRCN0000120450 Cct3-3 50.1 CCTCGAATTGTGCTATTGGAT TRCN0000120449 Cct4-1 44.5 CGGTTGCTGATGCTATTAGAA TRCN0000120456 Cct4-2 35.1 GCCCTTAGTGATCTTGCATTA TRCN0000120454 Cct4-3 32.1 GCGCAACTACTTCATTGAATT TRCN0000120453 Cct4-4 9.7 GCGGATGCTATGGAAGTCATT TRCN0000120455 Cct5-1 38.4 GCTGGGCTCCAAAGTGATTAA TRCN0000120435 Cct5-2 23.9 GCATGTCATAGAAACCTTGAT TRCN0000120436 Cct5-3 18.6 GCCACCTAAACCTAAGACAAA TRCN0000120432 Cct5-4 14.7 TGTCCATTTGAGCCACCTAAA TRCN0000120434 Cct6a-1 47.7 GCAGGGCTTGTCTATGAGTAT TRCN0000120443 Cct6a-2 22.1 CCTTCAGGAAACATTAGTTAA TRCN0000120444 Cct6a-3 11.1 CGTGTCCTTAGAGTATGAGAA TRCN0000120446 Cct6b-1 26.6 CCAAATAAGCATACTCTCATA TRCN0000120463 Cct6b-2 20.9 CGATAGCAGAAGCTCTTGTTA TRCN0000120462 Cct6b-3 17.8 CTCACCAAAGACGGCAATGTA TRCN0000120466 Cct7-1 50.1 GCCAAAGTCATCTTGTCTAAA TRCN0000120460 Cct7-2 41.9 GATGAGAAGATGGTTGCTGGT TRCN0000120457 Cct7-3 32.3 CGTGGATGAGACTATCAAGAA TRCN0000120459 Cct8-1 158.0 GCGGACATAGCTCTTCATTAT TRCN0000120476 Cct8-2 84.4 GCTGTAAAGGATATGTTAGAA TRCN0000120475 Cct8-3 42.8 CGGACCAAATGGAATGAATAA TRCN0000120473

shRNAs in bold were used in follow-up studies.

Slater et al. www.pnas.org/cgi/content/short/1302257110 6of6