WT

HrasG12V

WT+TPA

HrasG12V +TPA

HrasG12V

+TPA

H&E Ki67

Supplementary Fig 1: H&E and IHC staining for Ki67 in sections of mouse skin and papilloma from WT and HrasG12V mice treated with or without TPA: WT and HrasG12V mice were treated with TPA twice-weekly for 15 weeks. Sections of skin and papilloma tissues were stained with H&E (left) or by IHC for Ki67 (right). TPA induced hyperplastic changes in the epidermis and typical papillomas associated with increased Ki67 labeling in HrasG12V mice.

T G G T G G T G G G C G C T G G A G G C G T G G G A A A G A G T G C

T G G T G G T G G G C G C T G G A G G C G T G G G A A A G A G T G C67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

2225

0

Fragmenthras2_hrasseq

C T T A G A C A C A G C A G G T C T A G A A G A G T A T A G T G C C A

C T T A G A C A C A G C A G G T C T A G A A G A G T A T A G T G C C A421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

2225

0

Fragmenthras2_hrasseq

G T G G T G G T G G G C G C T G T A G G C G T G G G A A A G A G T G

G T G G T G G T G G G C G C T G T A G G C G T G G G A A A G A G T G66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

2232

0

Fragmenthras6_hrasseq

T T A G A C A C A G C A G G T C A A G A A G A G T A T A G T G C C A

T T A G A C A C A G C A G G T C A A G A A G A G T A T A G T G C C A422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

2232

0

Fragmenthras6_hrasseq

C T G G A G G G T C T A G A

Codons 12

Allele 1

C T G T A G G

G T C A A G A

Allele 2

A T

G T

Codons 61

Supplementary Fig 2: G12V and Q61L mutations are located on different Hras alleles in tumors harboring mutations at both sites: PCR was performed to amplify genomic DNA with primers bracketing the region encoding the G12V and G61L mutations of Hras from three papilloma DNAs harboring mutations at both sites (see lanes with asterisk in Fig 2). Products were then subcloned into pcDNA3 and sequenced. A representative trace of two clones from the same tumor shows two Hras alleles, each containing one of the mutations.

Normal

hyperplasia

papilloma

Before After Capped

Supplementary Fig 3: Laser-capture microdissection of mouse skin tissues: Frozen sections were subjected to laser-capture microdissection to isolate histologically normal, hyperplastic and papilloma cells for analysis of Hras allelic fractions (see Fig 2D). A representative photomicrograph before and after microdissection as well as of the capped tissues is shown.

WT HrasG12V papilloma

-Gsu

I

+G

su I

-Gsu

I

+G

su I

-Gsu

I

+G

su I

0

10

20

30

40

50

60

70

80

90

100

Hra

s m

RN

A l

eve

ls (

% o

f u

ncu

t)

- + - + - + - + - + - + - + - + - +

WT HrasG12V WT HrasG12V papillomas

Gsu I

A

B

Supplementary Fig 4: HRAS allelic imbalance in papilloma correlates with mRNA expression of HRAS: A) RT-PCR products from RNA isolated from 5 different papillomas from TPA treated HrasG12V mice as well as RT-PCR of RNA isolated from two WT and two HrasG12V MEFs were incubated with or without GSU I, which digests only WT HRAS cDNA. B) Quantitation of copy number data from the above experiments. Total HRAS cDNA in the absence of GSU I was normalized to 100%.

G T G G T G G T G G G C G C C G T C G G T G T G G G C A A N A G T

G T G G T G G T G G G C G C C G T C G G T G T G G G C A A N A G T42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

2751

0

Fragmentt24hrase1f-hhrase1f

G T G G T G G T G G G C G C C G G C C G T G T G G G C A A G A G T G

G T G G T G G T G G G C G C C G G C C G T G T G G G C A A G A G T G41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

3145

0

Fragmentc643hrase1f-hhrase1f

C643 (G13R) Hth83 (Q61R) T24 (G12V)G G N NN T N T N N T C C T C N N N N C C G N N N N N N N C NN G G

G G N NN T N T N N T C C T C N N N N C C G N N N N N N N C NN G G9 10 11 1213 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 3940 41 42

4033

0

Fragment17279-1-hrase2r2

A

- + - + - + - +EagIWT C643 WT Hth83B

C

WT C643 HTH830

102030405060708090

100110120 -Eag I

+Eag I

Cell line

Hra

s m

RN

A l

ev

els

(%

of

un

cu

t)

Supplementary Fig 5: HRAS allelic imbalance in human cancer cell lines with HRAS mutations: A) HRAS gene sequence traces from cancer cell lines with HRAS mutation: C643 (G13R, thyroid), Hth83 (Q61R, thyroid) and T24 (G12V, bladder). HRAS mutant peak is higher than WT allele in C643 and Hth83 cells. T24 has HRAS G12V homozygous mutation. B) PCR products from DNA isolated from C643 or Hth83 cells were incubated with or without EagI, which digests only mutant HRAS DNA. C) Quantitation of HRAS alleles from the above experiments. Total HRAS DNA in the absence of EagI was normalized to 100%. Data represents mean + SEM of 3 independent experiments.

Cal-62 Hth83

HRAS/KRAS

pMEKS217/221

-actin

KRAS

HRAS

Cal-62Hth-83

NRAS

pMEK

pERKT202/Y204

pAKTS473

actin

A

B S N H K S N H K

S 6 24 48 72 S 6 24 48 72

Supplementary Fig 6: Oncogenic RAS knockdown abrogrates MAPK signaling in cancer cell lines with RAS mutation. A) oncogenic Ras knockdown using RNAi in Hth-83 (HRAS mutation) and Cal62 ( KRAS mutation) cell lines for indicated time. Lysates were subjected to Western blotting with indicated antibodies. There are a marked decrease pMEK after oncogenic Ras knockdown over time in both cell lines. S: scramble B) isoform-specific Ras knockdown using RNAi in Hth-83 and Cal62 cells. Cell lystaes were subjected to Western blotting and detected with different antibodies. Only oncogenic Hras knockdown but not WT Kras and Nras diminished pMEK and pERK in Hth83 cells with Hras mutation. Only oncogenic Kras knockdown significantly diminished pMEK and pERK in Cal62 cells with Kras mutations. S:scramble, N:Nras, H:Hras, K:Kras. Presented data are representative for two independent experiments.

Cal62

0 1 2 3 4 50

200

400

600

800

1000

1200KRASHRASNRASScrambled

Day

Cel

ls/w

ell (

x103

)

HTH7

0 1 2 3 4 50

250

500

750

1000

1250

1500KRASHRASNRASScrambled

Day

Cel

ls/w

ell (

x103

)Hth83

0 1 2 3 4 50

500

1000

1500

2000

2500KRASHRASNRASScrambled

Day

Cel

ls/w

ell (

x103

)

Supplementary Fig 7: Oncogenic Ras knockdown inhibits cell proliferation. The indicated cell lines were transfected with indicated isoform-specific ras siRNA or scramble siRNA. Cells were counted at day 0 to day5. Each data point represents the mean plus SD of triplicates obtained in one of two independent experiments.

Cal62 Hth7

Hth83