% cells forming Neurospheres 81 ± 6 % 0 % 2.6 ± 0.7 % 76 ± 8 % 0 % 3.4 ± 0.6 % 83 ± 5 % 0 % 2.4 ± 0.9 % 89 ± 5 % 3 ± 1.5 % Total 10, ± 6 % 0 %
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1 Bo et l., Suppl. Tle 1 Supplementl Tle 1. Neurosphere formtion nd tumorigencity is enriched within the tumour cell popultions derived from humn primry glioms nd gliom xenogrfts. GBM smples or Gliom xenogrfts Cells % cells forming Neurospheres Minimum numer of cells for tumour initition (8 weeks) T3565 Primry Gliom CD ± 6 % % 5 No tumours detected t 2 x 1 6 Totl 2.6 ±.7 % 2, T3691 Primry Gliom CD ± 8 % % 1, No tumours detected t 2 x 1 6 Totl 3.4 ±.6 % 3, T375 Primry Gliom CD ± 5 % % 1, No tumours detected t 2 x 1 6 Totl 2.4 ±.9 % 4, 89 ± 5 % 3 D456MG CD133-3 ± 1.5 % Occ. smll tumours t 2 x 1 6 Totl 3.4 ±.6 % 1, 86 ± 6 % 5 D317 CD133- % No tumours detected t 2 x 1 6 Totl 3.8 ±.5 % 15, 81 ± 7 % 5 D54MG CD133- % No tumours detected t 2 x 1 6 Totl 2.9 ±.6 % 2,
2 Bo et l., Suppl. Tle 2 Supplementl Tle 2. In Vivo Limiting Dilution Tumorigenic Assys of Untreted nd Irrdited Cells GBM Smples Cell Type Minimum numer of cells for tumour initition (8 weeks) Untreted 2 Gy IR 5 Gy IR (Injected 48 hr fter IR) (Injected 48 hr fter IR) T3691 CD133- No tumours detected t 1 6 No tumours detected t 1 6 No tumours detected t 1 6 T3565 CD No tumours detected t 1 6 No tumours detected t 1 6 No tumours detected t 1 6 GBM Smples And Cell Type Untreted Minimum numer of cells for tumour initition (8 weeks) 24 hr fter 2 Gy IR 48 hr fter 2 Gy IR 72 hr fter 2 Gy IR 96 hr fter 2 Gy IR T T
3 Bo et l. Suppl Fig. S1 Untreted 48 hr fter IR (3 Gy) CD133- cells from D456MG CD133- cells from T367 GBM
4 Bo et l. Suppl Fig. S2 D54MG or D456MG IR Cell Deth D54R1 or D456R1 IR Cell Deth D54R2 or D456R2 IR Cell Deth D54R3 or D456R3 Cells (%) D54MG D456MG Prentl Lines Rdioresistnt Sulines
5 Bo et l., Suppl. Fig. S3 Cells (%) D456MG T3379X Untreted Xenogrft Irrdited Xenogrft (3 Gy) Dys until development of neurologic signs D456MG T3379X Mice injected with unfrctionted vile cells from untreted xenogrfts Mice injected with unfrctionted vile cells from In vivo irrdited xenogrfts
6 Bo et l., Suppl. Fig. S4 3B4-FITC Stining specific for humn cells confirmed humn origin of tumour cells isolted from gliom xenogrfts Negtive Control.49% 3B4-FITC Positive CD133- cells from D456MG Xenogrfts 99.47% 3B4-FITC Positive CD133- cells from D54MG Xenogrfts 99.1% 3B4-FITC Positive
7 Bo et l., Suppl. Fig. S5 FISH: Chromosome 1 Centromere Proe (Green) Control Norml cells T3359 Gliom Tumour cells
8 c Bo et l., Suppl. Fig. S5 T3359 CD133- gliom cells d T3359 CD133- gliom cells e T3359 CD133- gliom cells
9 Bo et l., Suppl. Fig. S6 Differentition of Cncer Stem Cells from D456MG Xenogrft Astrocyte linege GFAP S1 Oligodendrocyte linege O4 GlC Neuronl linege Mp2 TUJ1 Differentition of Cncer Stem Cells from 375 Primry Gliom Astrocyte linege GFAP S1 Oligodendrocyte linege O4 GlC Neuronl linege Mp2 TUJ1
10 Bo et l., Suppl. Fig. S7 Differentition of Untreted Cncer Stem Cells from 3781 Primry Gliom Astrocyte linege GFAP S1 Neuronl linege Mp2 TUJ1 Oligodendrocyte linege O4 GlC Differentition of Irrdited (2Gy) Cncer Stem Cells from 3781 Primry Gliom Astrocyte linege GFAP S1 Neuronl linege Mp2 TUJ1 Oligodendrocyte linege O4 GlC
11 Bo et l., Suppl. Fig. S8 Control IR (5 Gy) DBH + IR DBH (3 μm) D54MG CD133- In vivo irrditied xenogrfts C IR(2Gy) IR(5Gy) CD133: Cleved Cspse-3 D456MG Tuulin
12 D54MG Gliom Cell Survivl in Neurosl Medi without Growth Fctors 12 Cells CD133- Cells Bo et l., Suppl. Fig. S9 Cell Survivl (%) Dy1 Dy2 Dy3 Dy4 Dy5 Untreted Dy1 Dy2 Dy3 Dy4 Dy5 Irrdited (3 Gy) T3781 Primry Gliom Cell Survivl in Neurosl Medi without Growth Fctors 12 Cells CD133- Cells Cell Survivl (%) Dy1 Dy2 Dy3 Dy4 Dy5 Dy1 Dy2 Dy3 Dy4 Dy5 Untreted Irrdited (3 Gy)
13 Bo et l., Suppl. Fig. S1 Apoptotic Cell Deth of CD133- nd T375 Cells Induced y IR in Neurosl Medi with nd without Growth Fctors Cells CD133- Cells Annexin V-FITC Positive Cells (%) Untreted IR (3Gy) Untreted IR (3Gy) + EGF/FGF - EGF/FGF
14 Bo et l., Suppl. Fig. S11 Annexin V-FITC Mesurement of IR-induced Apoptotic Cell Deth of nd CD133- Cells from T3781 Primry Gliom Untreted IR (3 Gy) CD133- Annexin-V-FITC Positive Cells ( % ) cells Untreted CD133- cells IR(3Gy)
15 Bo et l., Suppl. Fig. S12 Gliom Cells from D456MG Control IR (5Gy) Dy 1 Dy 3 Dy 6 Dy 1 Dy 4 Dy 8 Control IR (5Gy) c Cells Derived from Cells (%) D456MG Untreted Irrdited Dy 1 Dy 4 Dy 8
16 Bo et l., Suppl. Fig. S13 2 Gy Irrdited cells derived from D456MG xenogrfts nd primry gliom specimens re cple of tumour initition in nude mouse rins Dys until development of neurologic signs D456MG T3359 T3379 Mice trnsplnted with untreted cells Mice trnsplnted with irrdited cells Purified surviving cells from 3 Gy irrdited D456MG nd T3379 Xenogrfts re cple of tumour initition in nude mouse rins Dys until development of neurologic signs D456MG T3379X Mice trnsplnted with cells from untreted xenogrfts Mice trnsplnted with vile cells from irrdited xenogrfts c Untreted CD133- Irrdited CD133- D456MG
17 Bo et l., Suppl. Fig. S14 D54MG T3359 CD NCS (1 ng/ml) patm (S1981) ATM (Totl) prd17 (S645) Rd17 (Totl) pchk1 (S345) Chk1 (Totl) pchk2 (S19) Chk2 (Totl) Tuulin CD NCS (1ng/ml) patm (S1981) ATM (Totl) prd17 (S645) Rd17 (Totl) pchk1 (S345) Chk1 (Totl) pchk2 (S19) Chk2 (Totl) Tuulin c ATM kinse ctivity induced y rdition in cells is higher thn in CD133- cells derived from humn primry gliolstom T3618 T3618 GBM CD133- IR (3Gy): prd17 (S645) Rd17-GST Input Tuulin (lystes for ATM IP)
18 Bo et l., Suppl. Fig. S15 D456MG CD133 - Cells CD133 + Cells Time After 3 Gy IR Tretment (hr) Untreted Cells With Comet's Til After IR (%) 12 CD133- Cells Cells D456MG Untreted Time After 3 Gy IR hr
19 CD133- Cells D456MG Bo et l., Suppl.Fig. S16 Cells Untreted 1 hr fter IR (3 Gy) 24 hr fter IR (3 Gy) Cells with p-h2ax Nucler Foci (%) CD133- Cells Cells Untreted 1 hr fter IR 24 hr fter IR
20 Bo et l., Suppl. Fig. S17 Untreted IR (5 Gy) DBH + IR DBH (3 μm) c Numer of Cncer Cells After Sorting (X1,) Rtio of Tumor Cells Derived From / CD133- cells Untreted IR IR+DBH DBH IJ Untreted IR IR+DBH DBH Tumour cells derived from CMTPX-leled cells (Red) Tumour cells derived from CFSE-leled CD133- cells (Green)
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