Figure S1. (a) Western blot of purified recombinant truncated His-hTAF91-140 and His-Fiber as detected by anti-His antibody. His-hTAF91-140 was verified by anti-TAF9 antibody. (b) ELISA showed direct binding between biotinylated p53 peptide (with putative TAF9 binding motif) and purified recombinant His-hTAF91-140 , but not the recombinant His-Fiber protein. (c) Gli dependent transcription activity was significantly inhibited by knocking down TAF9 expression with TAF9 shRNA in NSCLC cell line A549.

His-Fiber

His-TAF9WB:

anti-His

anti-TAF9 His-TAF9

His-TAF9

His-Fibera

0

20

40

60

80

100

1

Ctrl shRNA TAF9 shRNA

Gli1 + TAF9 Gli2 + TAF9

Gli

BS-

luc

activ

ity (%

)

0

0.5

1

1.5

0.5 uM 2 uM

OD

405

nm His-TAF

His-Fiber

biotin-p53 peptide

c

b

Figure S2. Effect of FN1-8 on other transcription factors. (a) CREB and HIF binding sites-luciferase reporter activities in stable cell lines 293 and NIH3T3 (Panomics) were not affected by FN1-8 (10 and 20 µM, 1 day). (b) The SOCS3 promoter (with STAT3 binding sites, (He, et al. BBRC 2003)) activity in A549 cells was not affected by by FN1-8 (20 µM, 1 day). All the measured luciferase activities were normalized to pRL‑TK vector (Promega) activity. Results are means + S.D.

020406080

100120

CREB BS-Luc HIF BS-Luc

Luc

activ

ity (%

)

DMSO 10µM FN1-8 20µM FN1-8

0

20

40

60

80

100

DMSO FN1-8

SOC

S3 p

rom

oter

ac

tivity

(%)

a b

b

0

20

40

60

80

100

Live

cel

ls (%

)

DMSO 10 µM FN1-8 30 µM FN1-8

Normal muscle cells Normal renal cells

c

Figure S3. Effect of FN1-8 on survival in several types of human normal cells. (a) Normal lung cells and (b) Normal muscle and renal cells were treated with different doses of FN1-8 for 4 days and then analyzed by flow cytometry. (c) Normal skin fibroblast were treated with different doses of FN1-8 and analyzed by MTS assay (time course). All results are mean values ± S.D. (error bars).

0

20

40

60

80

100

DMSO10 µM FN1-820 µM FN1-8

Normal skin fibroblast

1 2 3 4 5 6Days

Live

cel

ls (%

)

020406080

100

DMSO 10 uM 30 uM

Live

cel

ls (%

)

Normal lung cells

FN1-8

a

DMSO DMSOFN1-8 FN1-8

Kidney cortex Kidney medulla

Lung

Spleen Liver

Small intestine Colon

Ear

c

Figure S4. Pharmacokinetic (PK) and toxicity studies of FN1-8 in mice. (a) PK study of FN1-8 in mice (n=3) by i.p. injection (30 mg/kg). Results are means±SD. (b), (c) and (d) are toxicity analyses of FN1-8 in mice harboring H460 tumors of the efficacy study described in Figure 6. Body weights of mice were monitored during the period of drug administration (b). Hematoxylin and eosin (H&E) staining of organs (c). Leukocytes (WBC: white blood cell, NE: neutrophil, LY: lymphocyte, MO: monocyte, EO: eosinophil, BA: basophil) from animals were collected and counted through a blood cell counter (d). Normal ranges (x103 counts/ul) for the leukocyte population are: WBC: 1.8-10.7; NE: 0.1-2.4; LY: 0.9-9.3; MO: 0-0.4; EO: 0-0.2; BA: 0-0.2.

0

2

4

6

8

WBC NE LY MO EO BA

Leukocyte population

Cou

nts

(103 /μ

l) FN1-8Vehicle

d

a

0.0

5.0

10.015.0

20.0

25.0

10 15 20 25Time (days)

Body

wei

gth

(g)

FN1-8Vehicle

b

i.p. injection

Con

cent

ratio

n (n

g/m

l)

Time (hrs)

Table S2. Primer sequences for semi-quantitative RT-PCR

Gene Primer Sequences Size (bp)

Gli1 F: 5’-TACTCACGCCTCGAAAACCT-3’R: 5’-GTCTGCTTTCCTCCCTGATG-3’

340

Gli2 F: 5’-CACCAACCAGAACAAGCAGA-3’ R: 5’-ACCTCAGCCTCCTGCTTACA-3’

195

Shh F: 5’-CAGTGGCCAGGAGTGAAACT-3’ R: 5’-CCAGGAAAGTGAGGAAGTCG-3’

380

Ptch1 F: 5’-CGCCTATGCCTGTCTAACCATGC-3’R: 5’-TAAATCCATGCTGAGAATTGCA-3’

448

GAPDH F: 5'-ATGGGGAAGGTGAAGGTCGG-3'R: 5'-GACGGTGCCATGGAATTTGC-3'

180

Table S1. Peptide sequences for ELISA

Protein Motif Peptide Sequences

Gli1-TAF9bd (NH2)-LDSLDLDNTQLDFVAILDEPQG-(COOH)

Gli2-TAF9bd (NH2)-VDSQLLEAPQIDFDAIMDDGDH-(COOH)

Mutant Gli2-TAF9bd (NH2)-VDSQLLEAPQIDADAIADDGDH-(COOH)

p53-TAF9bd (NH2)-DPSVEPPLSQETFSDLWKLLPE-(COOH)