Supporting Information
Bailis et al. 10.1073/pnas.1706665114
SI Materials and Methods
Cell Culture.
HCT-116 O and HCT-116 N cells were maintained as
described in
Materials and Methods
in the main text. NCI-
H23 cells engineered to express inducible shRNA to MLH1 were
maintained in RPMI media in the absence (MMR-proficient) or
presence (MMR-deficient) of 500 ng/mL doxycycline (Sigma)
as described previously (20). Ovarian cancer cell lines were
obtained from American Type Culture Collection (ATCC) and
cultured accorded using the recommended conditions.
Cell Assays.
HCT-116 O H2B-GFP and HCT-116 N H2B-GFP cells
were plated to Perkin-Elmer 96-well View Plates at 2,500 cells per
well and incubated overnight. Cells were then treated with 0.5
μ
M
Rh-PPO or left untreated. Triplicate samples were imaged every
3 h over 144 h, using an IncuCyte (Essen Bioscience). Nuclear
count was determined over time for each field of view that was
imaged. For cell viability assays, cells were plated to View Plates at
1,000
–
5,000 cells per well and incubated overnight. Cells were
then treated with Rh-PPO in a dose
–
response for 3 d. Cells were
fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100
in PBS, and then stained with DAPI (Sigma). The number of
nuclei per well was counted using a Cellomics ArrayScan (Thermo
Fisher) and used to calculate the percent viable cells relative to
the number of cells from untreated samples.
An ELISA was used to evaluate the effects of compounds on
transcription. HCT-116 N and HCT-116 O cells were seeded in
96-well plates at 4,000 cells per well and allowed 24 h to adhere.
After 24 h, cells were treated with Rh-PPO (0
–
1
μ
M final con-
centration) or cisplatin (0
–
10
μ
M final concentration) at 37 °C
under humidified atmosphere for 24 h. After the incubation
period, an equal volume of 2 mM EU in prewarmed media was
added to each well, to a final concentration of 1 mM EU per well.
EU pulse labeling was carried out at 37 °C under humidified
atmosphere for 1 h. After the labeling period, the media was
removed, and cells were fixed with 3.7% formaldehyde in PBS
(pH 7.2) at 50
μ
L per well for 30 min at room temperature. The
fixident was removed, and wells were washed 1
×
with 200
μ
LPBS
(pH 7.2). Cells were then permeabilized with 0.5% Triton
X-100 in PBS (pH 7.2) (50
μ
L per well for 15 min) and washed with
PBS. A working solution of the Clic
k-It reaction mixture (Thermo
Fisher) was prepared immediately before treatment while pro-
tected from light, according to the manufacturer
’
s instructions. The
click reaction was carried out at ambient temperature for 30 min in
the absence of light. The wells were aspirated and washed with
rinse buffer. The plates were prote
cted from light, a
nd fluorescence
readout of the wells was carried out on a Flexstation 3 Multi-Mode
microplate reader (495-nm exci
tation, 519-nm emission, 515-nm
automatic cutoff; top-read).
Indirect immunofluorescence was carried out as described in
Materials and Methods
in the main text. Cells were plated to 96-
well View Plates (Perkin-Elmer) and allowed to adhere overnight
before treatment with compounds in a dose
–
response. After 24 h,
cells were fixed in 4% formaldehyde, permeabilized with 0.5%
Triton X-100 in PBS, and then blocked with 1
×
blocking buffer
(Thermo Fisher). Cells were incubated with primary antibody to
phosphorylated Chk1 serine 345 (Cell Signaling) at 1:100 in
blocking buffer overnight at 4 °C, and then washed in PBS, in-
cubated with a secondary antibody, anti-rabbit Alexa Fluor 488
(Thermo Fisher) and DAPI at 1:1,000 in blocking buffer for
30 min, and washed again in PBS. Stained cells were stored cov-
ered with PBS before imaging with a Cellomics ArrayScan
(Thermo Fisher) to measure staining intensity and nuclear count.
At least three independent experiments, with duplicate samples,
were performed. Data were analyzed with Graph Pad (Prism). A
Student
’
s
t
test was used to assess statistical significance.
Bailis et al.
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Fig. S1.
Growth of HCT-116 O and HCT-116 N cells treated with Rh-PPO. (
A
) HCT-116 O H2B-GFP and HCT-116 N H2B-GFP cells that were untreated, or treated
with Rh-PPO in a dose
–
response, were imaged every 3 h over 6 d. The graphs show the number of cell nuclei in a single field of view over time for triplicate
samples. (
B
) Representative images of HCT-116 O and HCT-116 N cells over time are shown. (Scale bar, 100
μ
M.)
Fig. S2.
Rh-PPO preferentially inhibits growth of NCI-H23 cells that are MMR-deficient. NCI-H23 cell lines containing a doxycycline-inducible shRNA to
MLH1 were grown continuously in the absence or presence of 0.5
μ
M doxycycline, resulting in MMR-deficient and MMR-proficient isogenic cell lines, re-
spectively. Cells were incubated with Rh-PPO in a dose
–
response for 72 h and then cell viability was assessed by a nuclear count assay. The graph shows cell
viability for duplicate samples at each concentration.
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Fig. S3.
Rh-PPO preferentially inhibits growth of ovarian cancer cell lines that are MMR-deficient. Five MMR-deficient cell lines and five MMR-proficient c
ell
lines were treated with Rh-PPO in a dose
–
response for 72 h and then cell viability was assessed by a nuclear count assay. (
A
) The graphs show dose
–
response
curves for cell viability of duplicate samples of ovarian cancer cell lines treated with Rh-PPO. (
B
) Comparison of the mean EC
50
values for cell viability of MMR-
deficient and MMR-proficient ovarian cancer cell lines (from
A
).
Fig. S4.
Rh-PPO inhibits transcription. HCT-116 O and HCT-116 N cells were treated with Rh-PPO as indicated for 24 h, and cells were labeled with EU for 1 h
before collection. EU incorporation was measured using a Click-It assay with fluorescent readout. Representative data from duplicate samples are s
hown.
Fig. S5.
Rh-PPO treatment activates the Chk1 checkpoint protein kinase. HCT-116 O and HCT-116 N cells were treated with Rh-PPO or cisplatin in a dose
–
response for 24 h and then cells were stained for phosphorylated serine 345 of Chk1 and counterstained for DAPI. The graphs show the percentage of cells
from duplicate samples that stained positive for phosphorylated Chk1.
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