Supplementary Figure S1

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Charles Webb
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1 Supplementary Figure S1 A (i) pscn-ndtpa (ii) () SM(PEO) 6 Mal-PEO6- -ndtpa Anti-γH2AX (i) (ii) EDC, sulfo-nhs NH 2 -SH -SH EGF EGF-PEO 6 - -ndtpa Anti-γH2AX-PNE (i) (ii) pscn-ndtpa SANH Anti-γH2AX -ndtpa Ser-SH NaIO 4 O=SH SH- -ndtpa SMPT EGF -SS- -ndtpa Supplementary Figure S1. Schematic overview of the synthesis of (A) non-cleavable anti-γh2ax-pne and () cleavable anti-γh2ax-n-ss-e.

Supplementary Figure S2 A nd (%) 123 I-EGF bou 1 5 EGF Anti-γH2AX-PNE P =.6 1-2 1-1 1 1 1 1 2 1 3 Log concentration competitor (nm) bound (%) 123 I-anti-γH2AX 1 Anti-γH2AX Anti-γH2AX-PNE 5 p =.

2 Supplementary Figure S2 A nd (%) 123 I-EGF bou 1 5 EGF Anti-γH2AX-PNE P = Log concentration competitor (nm) bound (%) 123 I-anti-γH2AX 1 Anti-γH2AX Anti-γH2AX-PNE 5 p = Log concentration competitor (nm) C kda Supplementary Figure S2. (A) MDA-M-468 cultures were exposed to 123 I-EGF for 2 h plus increasing concentrations of EGF, anti-γh2ax-pne or anti-γh2ax-n-ss-e. () Extracts from irradiated, γh2ax-containing 231-H2N cells were coated onto a RIA plate and exposed to 123 I-anti-γH2AX for 2 h plus increasing amounts of anti-γh2ax, antiγh2ax-pne or anti-γh2ax-n-ss-e. For both (A) and () the relative amount of 123 I that bound to plates was determined and LogIC 5 values were calculated. Results are shown as the mean of three replicates ± SD (C) PAGE analysis of 1. unmodified antiγh2ax, 2. anti-γh2ax-pne and 3. anti-γh2ax-n-ss-e.

3 Supplementary Figure S3 A 3 Control + Glutathione 4 CPM (normalised ) 2 1 CPM (normalised d) I-EGF Cleaved product Fraction fraction C CPM (normalis sed) IEGF I-EGF +cleaved low MW product Supplementary Figure S3. (A) Anti-γH2AX-N-SS-( 123 I-EGF) was exposed to or 1 mm glutathione. Size exclusion chromatography was performed using a G5 sephadex minicolumn. The amount of 123 I in each fraction was measured. () 123 I-EGF was synthesized as described d in the methods section. G25 size exclusion chromatography h was then performed on 123 I-EGF and the cleaved product resulting from (A). (C) To demonstrate that EGF is released from rigg-n-ss-e, rigg-n-ss-e was exposed to glutathione and purified by G5 size exclusion chromatography. Then, EGFR positive MDA-M-468 cells were exposed to 123 I-EGF for 2 h at 4 C with or without addition of the low molecular weight peak product. locking of 123 I-EGF binding to MDA-M-468 cells show that at least some of the low MW fraction binds to EGFR.

4 Supplementary Figure S4 A 231-H2N 4h post IR DAPI AF555- anti-γh2ax-pne Merge 4 Gy 4h post IR 4 Gy DAPI AF555- anti-γh2ax-n-ss-e Merge Supplementary Figure S4: (A) 231-H2N cells were exposed for 4 h to AF555- labeled anti- H2AX-PNE (red), fixed, and mounted with DAPI (blue). () 231-H2N cells were exposed for 4 h to AF555-labeled anti- H2AX-N-SS-E (red), fixed, and mounted with DAPI (blue).

exposed for 1 h to AF555-labeled antiγh2ax-n-ss-e or rigg-n-ss-e, and irradiated (4 Gy) or sham

5 Supplementary Figure S5 4 h post IR AF555-anti-γH2AX-N-SS-E or DAPI γh2ax AF555-rIgG-N-SS-E Merge 4 Gy 24 h post IR rigg-n-ss-e Gy rigg-n-ss-e 4 Gy Supplementary Figure S5: SQ2b cells were exposed for 1 h to AF555-labeled antiγh2ax-n-ss-e or rigg-n-ss-e, and irradiated (4 Gy) or sham irradiated. After 4 or 24 h, cells were fixed, stained for γh2ax (green) and mounted with DAPI (blue).

Supplementary Figure S6 A ound (cpm) 15 1 5 1 2 3 4 111 In-EGF (nm) MDA-M-468 Gy, 1h 4 Gy, 1h Gy, 4h 4 Gy, 4h SQ2b

Supplementary Figure S6: (A) MDA-M-468, SQ2b or 231-H2N cells were exposed to IR (4 Gy) or sham-irradiated.

To evaluate EGFR expression, the amount of membrane-bound 111 In was determined.

6 Supplementary Figure S6 A ound (cpm) In-EGF (nm) MDA-M-468 Gy, 1h 4 Gy, 1h Gy, 4h 4 Gy, 4h SQ2b Gy 1h 4 Gy 1h Gy 4h 4Gy4h 4h 231-H2N Gy, 1h 4 Gy, 1h Gy, 4h 4 Gy, 4h DAPI EGFR Merge 231-H2N SQ2b MD DA-M-468 Supplementary Figure S6: (A) MDA-M-468, SQ2b or 231-H2N cells were exposed to IR (4 Gy) or sham-irradiated. After 1or 4h, cells were exposed for 2h at 4 C to increasing concentrations of 111 In-EGF. To evaluate EGFR expression, the amount of membrane-bound 111 In was determined. The number of EGFR/cell was unaltered by IR. () MDA-M-468, SQ2b and 231-H2N cells were fixed, permeabilized and stained for EGFR (adapted from Cornelissen et al. J Nucl Med 211; 52: ).

7 Supplementary Figure S7 -SS- -SS- A. EGFR binding. EGFR-mediated C. Reductive internalization cleavage D. Endosomal escape & nuclear localization E. Irradiation & γh2ax foci formation F. 111 In-anti-γH2AX-N binds to γh2ax focus G. 111 In decays H. More DNA damage and more γh2ax foci I. Cell death Supplementary Figure S7. Proposed mechanism of action of 111 In-anti-γH2AX-N-SS-E. (A) 111 In-anti-γH2AX-N-SS-E binds to EGFR on the tumor cell membrane. () This binding triggers EGFR-mediated endocytosis and 111 In-anti-γH2AX-N-SS-E becomes encapsulated in an endosome. (C) There, glutathione and reductive enzymes cleave the disulphide bond, to release 111 In-anti-γH2AX-N, which (D) due to the NLS sequence, escapes from the endosome and localizes in the nucleus, through importin-nls interaction. (E) γh2ax foci form following IR. (F) 111 In-anti-γH2AX-N binds to γh2ax foci. (G) As 111 In decays, it emits a shower of Auger electrons at the DS site, which (H) causes more DSs, and more γh2ax foci to be formed, eventually (I) leading to cell death.

8 Supplementary Figure S8 relat tive 123 I counts I-NLS + NaIO4 2h + NaIO4 24h fraction Supplementary Figure S8: 123 I-labeled NLS shows a single peak on P4 SEC (black curve). When 123 I-NLS was reacted with.8 mg/ml NaIO 4 for 2 h there was no formation of higher molecular weight fragments (grey curve). However, after 24 h, there was evidence of dimerization (red curve).

Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the

Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the prey clones identified in the yeast two hybrid screen.