Nature - USA (2019-07-18)

Letter reSeArCH

a

Co

nsensus Sequenc

e

LAPC4

LNCaP

C42B

22RV1

Sanger Sequencing Chromatographs - FOXA1 Exon2

WT

(Plasmid CDS)

WT/P358fs

(Class2 mutant)

WT/WT

WT/WT

WT/WT

FOXA1

Genotype:

genomic DNA

358aa codon

heterozygous deletion of ‘C’

Vinculin

FOXA 1

N-term

(CST)

FOXA 1

N-term

(High Exp.)

LNCaPLAPC4FOXA1_FLFOXA1_d168FOXA1_d358

HEK293

PNT2RWPE1LNCaPVCaPLAPC4C42B22RV1Du145PC3

FOXA1

N-term

To t H3

70

40

50

35

15

Size:

(KDa)

FOXA 1

C-term

(Thermo)

V5-tag

Vinculin

FOXA1_FLFOXA1_d168FOXA1_d358

140

100

25

3540

50

70

25

3540

50

70

Recombinant

Protein

(V5-tagged): Size:

KDa

HEK293

FOXA1 N-terminal Antibody:

Cell Signaling Technologies - CST

(Cat#: 58613)

FOXA1 C-terminal Antibody:

Thermo Fisher Scientic - Thermo

(Cat#: PA5-27157)

FOXA1

C-term

PNT2LNCaPLNCaP-ARVCaPLAPC4C42B22RV1DU145PC3

70

40

50

140

100

25

3540

50

70

Size:

KDa

25

3540

50

70

C42B Cells (WT/WT)

2601

31834

40799

FOXA1 N-term FOXA1 C-term

LAPC4 Cells (WT/P358fs)

10nM DHT for 3h

334

2823

46973

hg38chr19: 50,851,000 50,853,000 50,855,000

KLK3

2 _

0.05 _

2 _

0.05 _2 _

0.05 _

2 _

0.05 _

1 _

0.05 _

FOXA1

N-term

FOXA1

N-term

FOXA1

C-term

FOXA1

C-term

AR

LAPC4L

APC4 + ex

o.FOXA1-F

L-V5

60bp DNA element used for EMSA

FOXA1-FL TD FKHD RD

FOXA1-d358 TD FKHD

FOXA1-d168 TD

1aa1 68 2694 72aa

V5-tag

N-term Ab

(epitope:

centered at148aa)

C-term Ab

(epitope: 412-472aa)

6_ ZBTB10

0.05 _

0.05_3 _

0.05 _

+DHT(10nM for 3h)

Chr8:

3 _

0.05 _6 _

hg38 50,845,000 50,850,000 50,855,000 80,485,000

4 _

0.05 _

3 _

0.05 _

4 _

0.05 _

3 _

0.05 _

LAPC4 (FOXA1

WT/P358fs

)

KLK3

Chr19:

FOXA1

N-term

FOXA1C-term

FOXA1

N-term

FOXA1C-term

LAPC4

(WT/P358fs)

LNCaP

(WT/WT)

C42B

(WT/WT)

50,845,000 50,850,000 50,855,000

4 _

0.05 _

3 _

0.05 _

4 _

0.05 _

3 _

4 _

0.05 _

3 _

0.05 _

0.05 _

KLK3

Chr19:

LAPC4

(WT/P358fs)

LNCaP

(WT/WT)

(WT/WT)C42B

FOXA1 N-term

FOXA1 C-term

C-term

N-term

IB: FO

XA1

Nuclear IP:

LAPC4 (WT/P358fs)

Input 1%IgGC-term

70

50

40

70

50

40

Size

KDa

FOXA1-WT FOXA1-P358fs

FOXA1FOXA1N-term

Vinculin^140

LAPC4+FOXA1-WT

0

2

4

6

8

10

12

(^14) NoDox
Dox(1ug/ml)
HPRT1 FOXA1-5’
LNCaP+FOXA1-P358fs
0
2
4
6
8
HPRT1 FOXA1-5’
Relative expression Relative expression
NoDox
Dox(1ug/ml)
b
c
d
e
f gh
j
i k
Extended Data Fig. 5 | See next page for caption.