SUPPLEMENTARY INFORMATION

Size: px

Start display at page:

Download "SUPPLEMENTARY INFORMATION"

Milton Anthony
5 years ago
Views:

1 Synthesis of ONC-101 nd Derivtives: The imidzo-pyrzine Synthesis The synthesis of imidzo[1,2-]pyrzines is simple three-component-one-pot rection s shown in Scheme 1: Scheme 1: The synthesis of imidzo[1,2-]pyrzines 1

2 Multi-component rections re vlule rections in comintoril chemistry, llowing the construction of trget compounds with severl diversity elements in single chemicl step 1-3. The synthesis of imidzo-pyrzines, uses three components: pyrzine, n ldehyde nd n isonitrile. The synthesis of ny imidzo-pyrzine is limited y the vilility of the strting mterils nd their reltive rectivity. Even though ldehyds re commercilly ville in gret vriety, this is no longer true for pyrzines with R1 H or R1 CH3 (oth commercilly ville). Therefore other pyrzines hve to e synthezided y 3-step synthesis 4. In the following, generl procedure for the preprtion of ONC 101 nd its precursors is given 5 : 2-mino-3-methyl-pyrzine (1; R1=CH3) In n utoclve, 2-chloro-3-methyl-pyrzine (10.0 g, 77.8 mmol) ws dissolved in dry methnol (30 ml). mmoni gs (60 g) ws dded. The mixture ws heted to 150 C for 8 hours. (strt pressure: 10 r, end pressure: 90 r). fter cooling to room temperture, the mixture ws evported to rown solid, which ws dissolved in 1N hydrochloric cid (100 ml) nd wshed with dichloromethne. The queous lyer ws slowly poured on cold sturted queous mmoni (150 ml), then extrcted with dichloromethne (3 x 100 ml). The comined orgnic lyers were dried (N2SO4) nd evported. The product ws extrcted from the residul solid with hot cetone (200 ml). Evportion yielded 36 % of the product s yellow solid. 2

3 (3-Chloro-phenyl)-(8-methyl-2-phenyl-imidzo[1,2-]pyrzin-3-yl)-mine (4; ONC101- NX-1) 2-mino-3-methyl-pyrzine (109 mg, 1.0 mmol), enzldehyde (106 mg, 1.0 mmol) nd 3-chloro-phenylisonitrile (138 mg, 1.0 mmol) were dissolved in mixture of dry methnol (2.0 ml) nd trimethyl orthoformte (2.0 ml) under rgon. The mixture ws stirred t 60 C for 3 hours, then cooled to RT. n nlyticlly pure smple ws otined from the crude product using preprtive HPLC. 1 Prchinsky, V.Z. et l., ir-oxidized products of multi-component rections etween 3-mino-1,2,4-trizole, romtic ldehydes nd isonitriles. Tetrhedron Lett. 47, (2006). 2 Prchinsky, V.Z. et l., Multi-component rections etween 2-minopyrimidine, ldehydes nd isonitriles: the use of nonpolr solvent suppresses formtion of multiple products. Tetrhedron Lett. 47, (2006). 3 Msquelin, T. et l., Sequentil Ugi/Strecker rections vi microwve ssisted orgnic synthesis: novel 3-center-4-component nd 3-center-5-component multicomponent rections. Tetrhedron Lett. 47, (2006). 4 Krms, G. & Spoerri, P.E. The preprtion of hydroxypyrzines nd derived chloropyrzines. J. m. Chem. Soc. 74, (1952). 5 Berset, C. et l., ngiogenesis inhiitors. Ptent No. WO/2006/13003 (2006). 3

4 wild-type Efn2 KO VEGF VEGF ephrin-b2 VEGFR * * endocytosis impired VEGFR ctivtion nd endocytosis full VEGFR signling compromised signling * * * * VEGF endothelil sprouting VEGFR VEGFR endocytosis & signling motility invsive ctivity EphB? Supplementry Figure 1: Schemtic summry of findings., Ephrin-B2 is essentil for VEGF induced internliztion nd the full ctivtion (sterisks) of VEGF receptors. Both VEGFR endocytosis nd downstrem signl trnsduction re compromised in Efn2 knockout endothelil cells., Ephrin-B2 nd interctions with EphB receptors promote endothelil sprouting nd therey the ngiogenic growth of lood vessels. We propose tht ephrin-b2 nd its downstrem (reverse) signl trnsduction ctivity integrte VEGF signlling nd other Eph/ephrin regulted cellulr processes such s the of cytoskeletl orgnistion, motility, invsiveness nd dhesion. The exct contriution of EphB receptor tyrosine kinses nd their role in VEGFR endocytosis remin to e ddressed. 4

doi: 10.1038/nture09002 50 µm Supplementry Figures Supplementry Figure 1: Schemtic summry of findings.

Both VEGFR endocytosis nd downstrem signl 20 µm V V trnsduction re compromised in Efn2 knockout endothelil cells.

5 doi: /nture µm Supplementry Figures Supplementry Figure 1: Schemtic summry of findings. isolectin B4 Efn2-GFP isolectin B4 Efn2-GFP DPI, Ephrin-B2 is essentil for VEGF induced internliztion nd the full ctivtion (sterisks) of VEGF receptors. Both VEGFR endocytosis nd downstrem signl 20 µm V V trnsduction re compromised in Efn2 knockout endothelil cells., Ephrin-B2 nd interctions with EphB receptors promote endothelil sprouting nd therey the ngiogenic growth of lood vessels. We propose tht ephrin-b2 nd its downstrem (reverse) signl trnsduction ctivity integrte VEGF signlling nd other Eph/ephrin regulted cellulr processes such s the of cytoskeletl orgnistion, motility, invsiveness nd dhesion. The exct contriution of EphB receptor tyrosine kinses nd their role in VEGFR endocytosis remin to e ddressed. isolectin B4 EphB4 EphB4 Supplementry Figure 2: Ephrin B2 nd EphB4 expression in retinl ECs., Whole mount GFP (green) nd isolectin B4 (ib4, red) stining in the P5 Efn2 GFP centrl retinl vsculture ehind the edge of the growing endothelil plexus.2, Positive Suppl. Figure Wng et l. cells re found in rteries (), cpillries nd non-vsculr cells. Nuclei, TO PRO3 (lue)., Whole mount nti EphB4 immunofluorescence predominntly lels veins (V) ut lso cpillries in centrl region of the P5 wild type retin. Supplementry Figure 3: Inducile gene trgeting in the vsculr system. 4 5

Eph/ephrin regulted cellulr processes such s the of cytoskeletl orgnistion, motility, invsiveness nd dhesion.

ECs., Whole mount GFP (green) nd isolectin B4 (ib4, red) stining in the P5 Efn2 GFP centrl retinl vsculture

Positive cells re found in rteries (), cpillries nd non-vsculr cells. Nuclei, TO PRO3 (lue).

region of the P5 wild type retin. Efn2 iδec Supplementry Figure 3: Inducile gene trgeting in the vsculr system.

6 doi: /nture09002 skin mesentery 500µm L C V Supplementry Figures hind lim 500µm C skin 500µm 500µm L V V L Supplementry Figure 1: Schemtic summry of findings. C, Ephrin-B2 is essentil for VEGF induced internliztion nd the full ctivtion intestine (sterisks) of VEGF receptors. Both VEGFR endocytosis ovrynd downstrem signl Peyer s ptch uterus 500µm 500µm trnsduction re compromised in Efn2 knockout endothelil cells. 500µm 500µm, Ephrin-B2 nd interctions with EphB receptors promote endothelil sprouting nd therey the ngiogenic growth of lood vessels. We propose tht ephrin-b2 nd its downstrem (reverse) signl trnsduction ctivity integrte VEGF signlling nd other Eph/ephrin regulted cellulr processes such s the of cytoskeletl orgnistion, motility, invsiveness nd dhesion. The exct contriution of EphB receptor tyrosine c isolectin B4 d kinses nd their role in VEGFR endocytosis remin to e ddressed. R26-YFP Cre reporter ephrin-b2 α-sm 100 µm 5µm Supplementry Figure 2: Ephrin B2 nd EphB4 expression in retinl ECs., Whole mount GFP (green) nd isolectin B4 (ib4, red) stining in the P5 Efn2 GFP centrl retinl vsculture ehind the edge of the growing endothelil plexus. Positive cells re found in rteries (), cpillries nd non-vsculr cells. Nuclei, TO PRO3 (lue). 5µm retin, Whole mount nti EphB4 immunofluorescence predominntly lels veins (V) ut lso cpillries in centrl region of the P5 wild type retin. Efn2 iδec Supplementry Figure 3: Inducile gene trgeting in the vsculr system., β glctosidse stined E18.5 tissues isolted from Cdh5 CreERT2 trnsgenics in the Suppl. Figure 3, Wng et l. ROS26R Cre reporter ckground., rteries; V, veins; C, cpillries; L, lymphtics. 4, ROS26R Cre reporter ctivtion in rnge of Cdh5 CreERT2 trnsgenic dult tissues, s indicted. c, ROS26 YFP Cre reporter (green) expression in the P5 Cdh5 CreERT2 retinl vsculture. EC were lelled with isolectin B4 (lue). d, Downregultion of ephrin-b2 protein (green) in the endothelium of derml lood vessels t E µm thick prffin sections re shown. Vsculr smooth muscle cells re lelled y α-smooth muscle ctin (red). Supplementry Figure 4: Defective endothelil sprouting in the Efn2iΔEC retin. Shown re representtive imges of P6 Efn2iΔEC mutnts generted with the Cdh5 CreERT2 6

7 Efn2 i EC (Cdh5 CreERT2) 100 µm 100 µm 25 µm 25 µm Supplementry Figure 4: Defective endothelil sprouting in the Efn2 iδec retin. Shown re representtive imges of P6 Efn2 iδec mutnts generted with the Cdh5 CreERT2 trnsgenic line. Note impired vsculr growth nd reduced rnching. ECs were lelled with isolectin B4. 7

efn2 MO-1 efn2 MO-2 20µm Ephrin-B2 MO efn2 MO c

Supplementry Figure 5: efn2 knockdown in zerfish.

the dorsl ort (rrows) nd somtic cells in zerfish

Ephrin B2 expression in these domins ws strongly

showing intersegmentl sprouts of nd efn2

Control nd lunt morphnt filopodi (rrows) re mrked.

8 efn2 MO-1 efn2 MO-2 20µm Ephrin-B2 MO efn2 MO c 200µm 50µm 15µm 15µm fli-egfp Efn2 KO 200µm 50µm Supplementry Figure 5: efn2 knockdown in zerfish., nti ephrin B2 ntiody stining of sections lels the dorsl ort (rrows) nd somtic cells in zerfish emryos (24hpf). Ephrin B2 expression in these domins ws strongly reduced fter injection of two different efn2 morpholinos (MO-1 nd MO-2)., Still imges from Supplementry Movies 1 nd 2 showing intersegmentl sprouts of nd efn2 knockdown fli-egfp zerfish emryos (27hpf). Control nd lunt morphnt filopodi (rrows) re mrked. c, Network formtion y cultured mouse nd Efn2 KO ECs. The ltter lck cellulr protrusions (rrows). Phlloidin (green) lels F-ctin. 8

75 50 igof IP ephrin-b2 WB ephrin-b2 ephrin-b2 α SM V L Efn2

Supplementry Figure 6: Inducile overexpression of ephrin-b2

, Immunoprecipittion (IP) nd Western lot (WB) of ephrin B2

, Immunofluorescence on tissue sections of or Efn2 igof

9 75 50 igof IP ephrin-b2 WB ephrin-b2 ephrin-b2 α SM V L Efn2 igof 20µm V 20µm L c Efn2 igof 20µm 20µm endomucin Supplementry Figure 6: Inducile overexpression of ephrin-b2 in ECs., Immunoprecipittion (IP) nd Western lot (WB) of ephrin B2 (~45kD) nd the CFP ephrin B2 fusion protein (75kD) from Efn2 igof (Tie2-rtT x teto-efn2) nd tissues. Moleculr weights (kd) re indicted., Immunofluorescence on tissue sections of or Efn2 igof mesentery with indicted ntiodies. Overexpression of ephrin-b2 in mutnt rteries (), veins (V) nd lymphtic vessels (L). c, Whole mount stining of nd Efn2 igof derml lood vessels t E

10µm iδec igof 10µm PECM1 collgen IV 10µm 10µm PECM1

ctrl iδec col IV sleeves/field 5 4 3 2 1 0 P=0,0013

rrows mrk sprouts, rrowheds empty collgen IV sleeves.

non-retrcted (PECM1+ collgen IV+) sprouts in the E18.

10 10µm iδec igof 10µm PECM1 collgen IV 10µm 10µm PECM1 collgen IV c retrcted tips (%) P=0,0004 ctrl iδec col IV sleeves/field P=0,0013 ctrl igof Supplementry Figure 7: Ephrin-B2 nd endothelil sprouting. nd, PECM1 (green) nd collgen IV (red) stining of whole mount E15.5 () nd E18.5 () nd mutnt skin smples s indicted. rrows mrk sprouts, rrowheds empty collgen IV sleeves. c, Rtio of retrcted (PECM1- collgen IV+) vs. non-retrcted (PECM1+ collgen IV+) sprouts in the E18.5 Efn2 iδec derml vsculture (left). Quntittion of empty collgen IV sleeves (corresponding to ndoned sprouts nd lost vsculr connections) in Efn2 igof mutnts. P vlues (c) were clculted using two-tiled Student s t-test. Error rs, s.e.m. n 3. 10

11 100µm 100µm collgen IV 10µm 10µm Efn2 igof 10µm collgen IV Efn2 igof Efn2 igof 10µm 10µm Efn2 igof Efn2 iδec c 2µm 2µm 2µm Efn2 igof Efn2 iδec 2µm 2µm 2µm Efn2 igof Efn2 iδec Supplementry Figure 8: Morphology of Efn2 mutnt mtrix sleeves nd ECs. vsculture. Note undnce of thin mtrix structures in the mutnt vsculture (rrowheds)., Confocl imges of collgen IV+ structures in the E18.5 derml vsculture. Long, sprout like mtrix sleeves (rrows) nd thin mtrix connections (rrowhed) re chrcteristic for Efn2 igof mutnts. Collgen IV sleeves re short nd lunt in Efn2 loss-of-function mutnts. c, Electron microgrphs of E18.5 nd Efn2 mutnt derml cpillries, s indicted. Efn2 igof ECs re thin nd highly ruffled wheres Efn2 iδec cpillry ECs re thick nd devoid of protrusions. 11

Figure 9:, Ephrin-B2 overexpression in individul ECs (sterisk) within HUVEC monolyer.

12 15µm microinjection * * migrtion speed (µm/min) 2,5 2 1,5 1 0,5 0 vector ephrin-b2 microinjected vehicle ONC-102 ONC-101 totl distnce (µm) vector ephrin-b2 microinjected vehicle ONC-102 ONC-101 Supplementry Figure 9:, Ephrin-B2 overexpression in individul ECs (sterisk) within HUVEC monolyer. Shown re still imges of long endothelil processes (rrows) from Movie S3., Quntittion of migrtion speed nd distnce sed on 8 independent microinjection experiments ech. 12

Kinse IC 50 enzyme ssy K d ffinity ssy IC 50 enzyme ssy K d ffinity ssy Kinse [μm] [μm] [μm] [μm] Eph1 0.19 0.89 FGFR3K > 10 > 10 Eph2 0.63 2.1 GSK3α n/ > 10 Eph3 n/ 3.6 GSK3β n/ > 10 Eph4 n/ 1.

64 KIT > 10 > 10 EphB4 0.33 0.41 LCK > 10 3.8 BRF 0.70 0.58 MEK1 n/ 2.4 BRF V600E 0.87 0.77 MEK2 n/ 5.2 CRF1 0.46 0.44 MERTK n/ 3.4 EGFR 0.55 0.95 MET > 10 > 10 EGFR(747-752del) n/ 1.

13 Kinse IC 50 enzyme ssy K d ffinity ssy IC 50 enzyme ssy K d ffinity ssy Kinse [μm] [μm] [μm] [μm] Eph FGFR3K > 10 > 10 Eph GSK3α n/ > 10 Eph3 n/ 3.6 GSK3β n/ > 10 Eph4 n/ 1.3 HCK > 10 > 10 Eph5 n/ 3.3 IGF1R > 10 > 10 Eph INS1R > 10 > 10 Eph7 n/ 29 JK1 > 10 > 10 Eph8 n/ 6.4 JK2 > 10 > 10 EphB JK3 > 10 > 10 EphB KDR > 10 > 10 EphB KIT > 10 > 10 EphB LCK > BRF MEK1 n/ 2.4 BRF V600E MEK2 n/ 5.2 CRF MERTK n/ 3.4 EGFR MET > 10 > 10 EGFR( del) n/ 1.3 MK2 > 10 > 10 EGFR(L858R) n/ 1.1 PDGFRα > 10 > 10 EGFR(L861Q) n/ 0.88 PDK1 > 10 > 10 ERBB2 n/ > 10 PK > 10 > 10 ERBB4 n/ 3.8 PKBα > 10 > 10 BL RET > 10 > 10 LK > 10 > 10 ROCK > 10 > 10 CDK2 > 10 > 10 SYK > 10 > 10 CK1α SRC > 10 > 10 FGFR-4 > 10 > 10 TYK2 > 10 > 10 20µm p-eph EphB4 c EphB4 inj. 20µm EphB4 inj. + ONC-102 p-eph dextrn ephrin-b2 inj. ephrin-b2 inj. + ONC-101 ephrin-b2 inj. + ONC-102 Supplementry Figure 10: Chrcteristion of Eph kinse inhiitors., ctivity nd inding profiles (IC 50 nd K d ) of ONC 101., Detection of EphB4 (red) nd phosphorylted Eph receptor (green) in microinjected HUVECs overexpressing EphB4. Specific phospho Eph (p Eph) signls re sent fter tretment with ONC 102 (10µM). c, Eph tyrosine phosphoryltion (green, rrows) in HUVECs surrounding microinjected (dextrn contining) ephrin-b2-overexpressing cells. No p Eph signl ove ckground level cn e seen fter tretment with ONC 101 or ONC

14 Chrcteriztion of ONC-101 Physicochemicl properties MW: g/mol ppernce: light rown solid HPLC: Purity: 254 nm Method: 1% cetonitrile to 100% in 10 min, 0.1% TF Flowrte: 1.0 ml/min ColmMn: HP Hypersil BDS C18, 125 * 4 mm Retention: min 1H-NMR: consistent 13C-NMR: consistent MssSpec: consistent (ESI) Melting Point: N/ LOGP: 4.42 Soluility: Kinse uffer (Upstte), 1% DMSO: ~100 μm Kinse uffer (Upstte), 0.25 % DMSO: ~65 μm NMR nlysis: Supplementry Figure 11: Chrcteristion of ONC 101. Physicochemicl properties, the chemicl structure of ONC 101 nd the NMR nlysis confirming the chemicl identity of the compound re shown. 14

15 Chrcteriztion of ONC-101: HPLC nd mss spectroscopic nlysis Supplementry Figure 12: Chrcteristion of ONC 101. HPLC nd mss spectroscopic dt confirming the purity of ONC 101 re shown. 15

16 Chrcteriztion of ONC-102 Physicochemicl properties MW: g/mol ppernce : light rown solid HPLC: Purity: 254 nm Method: 1% cetonitrile to 100% in 10 min, 0.1% TF Flowrte: 1.0 ml/min Column: HP Hypersil BDS C18, 125 * 4 mm Retention: min 1H-NMR: consistent 13C-NMR: consistent MssSpec: consistent (ESI) Melting Point: N/ LOGP: 4.71 Soluility: Kinse uffer (Upstte), 1% DMSO: ~100 μm Kinse uffer (Upstte), 0.25 % DMSO: ~50 μm NMR nlysis: Supplementry Figure 13: Chrcteristion of ONC 102. Physicochemicl properties, the chemicl structure of ONC 102 nd the NMR nlysis confirming the chemicl identity of the compound re shown. 16

17 Efn2 lox/lox (P14 skin) Efn2 i EC (P14 skin) 100µm grtion (%) LYVE1 α-sm Supplementry Figure 14: Ephrin-B2 nd lymphngiogenesis. Reduced growth of LYVE1-positive (green) derml lymphtic vessels in postntlly induced P14 Efn2 iδec mutnts. 17

18 Efn2 KO EGF: surfce EGFR tuulin VEGF-C: p-erk1/2 ERK1/2 Efn2 KO c VEGF-C: p-kt kt Efn2 KO d 250 e cell migrtion (%) cell migrtion (%) cell migrtion (%) unstim. VEGF VEGF C unstim. VEGF VEGF C 50 0 unstim. VEGF C ngii FGF EGF unstim. VEGF C ngii FGF EGF ECs Efn2 KO ECs VEGF- VEGF-C VEGF- VEGF-C ECs Efn2 KO ECs Supplementry Figure 15: Loss of ephrin-b2 ffects VEGF-C induced signlling nd chemotxis., Detection of surfce EGFR in stimulted nd Efn2 KO ECs y iotinyltion.,c, VEGF C induced kt nd ERK1/2 ctivtion is compromised in Efn2 KO ECs. d, Efn2 KO ECs show compromised VEGF or VEGF C induced migrtion in Boyden chmer ssys. e, Loss of ephrin-b2 selectively impirs chemotxis induced y VEGF C ut not ngiotensin II (ng II) or firolst growth fctor (FGF). Efn2 KO ECs show enhnced chemotxis towrds epiderml growth fctor (EGF). 18

PROX1 10 µm d, Efn2 KO ECs show compromised VEGF or VEGF C induced migrtion in Boyden chmer ssys.

Efn2 KO ECs show enhnced chemotxis towrds epiderml growth fctor (EGF). Supplementry 16: VEGFR3 internlistion in Efn2 mutnts.

mesenteric lymphtic vessels fter injection of PBS ( for the experiment shown in Figure 4). Nuclei, DPI (lue).

19 doi: /nture09002 Efn2lox/lox (P6 mesentery) Efn2i EC (P6 mesentery) 15 µm 15 µm PBS VEGFR3 DPI Efn2lox/lox (P6 mesentery) Efn2i EC (P6 mesentery) 10 µm VEGFR3 PROX1 10 µm d, Efn2 KO ECs show compromised VEGF or VEGF C induced migrtion in Boyden chmer ssys. cell migrtion (%) e, Loss of ephrin-b2 selectively impirs chemotxis induced y VEGF C ut not ngiotensin II (ng II) or firolst growth fctor (FGF). Efn2 KO ECs show enhnced chemotxis towrds epiderml growth fctor (EGF). Supplementry 16: VEGFR3 internlistion in Efn2 mutnts. VEGF- VEGF-C Figure VEGF- VEGF-C, Confocl imges of VEGFR3 (green) distriution in P6 nd Efn2iΔEC Suppl. Figure 16, Wng et l. mesenteric lymphtic vessels fter injection of PBS ( for the experiment shown in Figure 4). Nuclei, DPI (lue)., Confocl imges of VEGFR3 distriution in P6 nd Efn2iΔEC mesenteric lymphtic vessels fter intrperitonel injection of VEGF C. ccumultion of VEGFR3 (green) occurs round Prox1-positive (red) ut not mutnt LEC nuclei. Supplementry Movies: S1, Fluorescent time-lpse movie showing dynmics of intersegmentl vessels in 27 hpf fli1-egfp emryo injected with morpholino (i.e., efn2-mo contining 5 point muttions). Numerous filopodi cn e seen on ngiogenic endothelil sprouts visulized y EGFP very similr to uninjected emryos (dt not shown). 19

20 Supplementry Movies: S1, Fluorescent time-lpse movie showing dynmics of intersegmentl vessels in 27 hpf fli1-egfp emryo injected with morpholino (i.e., efn2-mo contining 5 point muttions). Numerous filopodi cn e seen on ngiogenic endothelil sprouts visulized y EGFP very similr to uninjected emryos (dt not shown). S2, Intersegmentl vessels in 27 hpf efn2-mo-injected fli1-egfp emryo showed few filopodi nd insted lunt, le-like protrusions were seen on the cell surfce. Still imges from Movies S1 nd S2 t time points 0, 30, 60 minutes re shown in Supplementry Figure 4. ctul durtion of oth movies is 60 minutes with 61 frmes t 1 minute intervls. S3, Ephrin-B2 overexpression in single cells within confluent monolyer of (uninjected) HUVECs. The injected cells displyed incresed motility nd dynmic sprout formtion. Movie commences 5 minutes fter injection nd corresponds to 300 minutes (600 frmes). Selected still imges re shown in Supplementry Figure 8. 20

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:1.138/nture1794 BR EPFs BRI1? ERECTA TMM BSKs YDA PP2A BSU1 BIN2 pbzr1/2 BZR1/2 MKK4/5/7/9 MPK3/6 SPCH Cell growth Stomtl production Supplementry Figure 1. The model of BR nd stomtl signling pthwys.