figure s1: schematic for generation of bacterial artificial...
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Supplemental Figure S1
pLD53.SCA-E-B
GFP
Am
p Rec
A
SacB omp R6Kr
Chga 5’ Chga 3’
Chga 5’ Chga 3’
BAC Chga-GFP transgene
Homologous
recombination
exon 3
GFP
exon 1
Chga locus (11.5 kb)
105 kb downstream
89 k
b up
stre
am
Chloramphenicol
exon 1 exon 2
Chga locus (11.5 kb)
BAC vector (RP23-377G8)
Chloramphenicol
exon 3 105 kb dow
nstream
89 k
b u
pstr
eam
Figure S1: Schematic for generation of bacterial artificial chromosome-based Chga-GFP transgene. Two segments of the mouse Chromogranin A (Chga) gene were amplified from BAC clone 377G8 using PCR and were subcloned into the pLD53.SC-AB plasmid. The resulting plasmid was transformed into a strain of E.coli containing the BAC 377G8, and recombinants were selected using chloramphenicol as a selection and sucrose for counterselection. This procedure generated a recombinant BAC (right) in which the GFP gene replaced part of exon 1 and all of exon 2 of Chga gene, but that still contained 89 kB sequence upstream and 105 kB sequence downstream of the transcriptional start site.
GFP Phase-contrast
Supplemental Figure S2
Cgrp
200 0
600 400
800
2
6
10
0
4
8
Nfib
5
0
15
10
E2f1 Syp
Rel
ativ
e Le
vel
50
15 10
20
200 0
400 600 800
Chga
Figure S2: A) Phase contrast and fluorescence images showing FACS-sorted GFP-positive cells 5 days after being isolated from lungs of Chga-GFP mice. Normal (arrows), potentially dying (arrowheads), and dead cells are apparent. B) RT-qPCR data showing expression of several neuroendocrine-specific markers and other oncogenes in normal lung (lung), preSC, and SCLC (n=3) relative to ARBP P0 control primers. C) preSCs infected with retroviruses expressing N-Myc, and different levels of c-Myc (n=3). D) Nude mice, 1 month after injection of cells infected with retro-GFP, retro-N-Myc, and retro-c-Myc. Arrow points to a tumor. Asterisk indicate an area of injection of control preSC. E) RT-qPCR data showing expression of the Myc family genes in the GFP control and Myc family member infected preSCs. Expression is relative to ARBP P0 F) CGRP immunohistochemistry showing neuroendocrine features of L-Myc-preSC allograft similar to SCLC allograft. Size bars: A and C, 10µm; D, 1cm.
GFP
N-Myc
L-Myc
60 40 20 0 100 50 0 4 3 2 1 0
c-Myclow
E Relative level Relative level Relative level
GFP-preSC c-Myc-preSC N-Myc-preSC D
CN-Myc c-Mychigh c-Myclow
B
L-Myc-preSC allograft SCLC allograft F
CGRP
A
*
CGRP
c-Myc-preSC N-Myc-preSC L-Myc-preSC
Supplemental Figure S3
Figure S3: A) PCR strategy to test for Mycl recombination (top) with PCR data from tumors and tails showing recombination of floxed Mycl allele in SCLC tumors. B) CGRP immunohistochemistry showing positive staining in Rb/p53/p130 and Rb/p53/p130/Mycl mutant SCLC. Scale bar = 200 microns. C) Real time PCR showing Mycl and Mycn copy number relative to control Ant1 gene. Data were normalized to normal tail DNA D) Sequencing traces showing CRISPR-mediated deletions of MYC members in mouse embryo fibroblasts.
L-Mycl lox/lox Mycl +/+
Rblox/lox; p53lox/lox; p130lox/lox B
CGRP
307 bp Exon1 Exon2 Exon3
floxed Mycl allele
wt Mycl allele
247 bp forward and reverse primers
A
Mycn
β-actin
Tail Lung tumors
Mycl 307bp 247bp
NW217T1
NW196T1
F861T1
F855T1
NW169T1
NW173T1
NW163T2
NW157T1
NW222T1
F643T1
F612T2
F658T1
F658T2
F862T1
NW483T1
NW149T1
NW224T1
F616T10
20
40
60
80
DNA
copy
num
ber
norm
alize
to ta
ils
Nmyc genomic DNA copy number
Rb/p53/Pten/Mycl Rb/p53/Pten
NW217T1
NW196T1
F861T1
F855T1
NW169T1
NW173T1
NW163T2
NW157T1
NW222T1
F643T1
F612T2
F658T1
F658T2
F862T1
NW483T1
NW149T1
NW224T1
F616T10
20
40
60
80
DNA
copy
num
ber
norm
alize
to ta
ils
Lmyc genomic DNA copy number
Rb/p53/Pten/Mycl Rb/p53/Pten
L-Myc copy number
N-Myc copy number
C
Mycn exon2
LacZ-CRISPR
MycN-CRISPR
Mycl exon1
LacZ-CRISPR
Mycl-CRISPR
Myc exon2
LacZ-CRISPR
Myc-CRISPR
Genomic regions targeted by guide RNA
D
loxP element
DN
A co
py n
umbe
r nor
mal
ized
to ta
ils
CGRP
Supplemental Figure S4
MYCL
Rel
ativ
e ex
pres
sion
leve
l (M
YCL/
AR
BP
P0)
150
50
0
200
100
250 MYCN
Rel
ativ
e ex
pres
sion
leve
l (M
YCN
/AR
PL P
0)
6
2 0
600
4
800
60
20
40
1000
Rel
ativ
e ex
pres
sion
leve
l (M
YC/A
RB
P P0
)
15
5
0
20
10
25 MYC
Figure S4: A) RT-qPCR data showing expression of MYC members relative to ARBP P0 across the indicated human SCLC cell lines. Levels are expressed relative to those in 293T cells B) Cleaved Caspase 3 (CC3) immunostaining (left) and quantification (right) in SCLC tumors from mice treated for 2 weeks with CX-5461 or in control untreated mice.
control CX-54610
2
4
6
Per
cent
age
of P
ositi
ve C
ells Cleaved Caspase 3
A
B
CC
3 st
aini
ng
% C
C3-
posi
tive
cells
Vehicle CX-5461 (2 weeks)
Vehicle CX-5461 (2 weeks)