Supplementary data not shown
Fig. S1: RT-PCR of CpdA and Dex repression of mouse CBG: BWTG3 cells were induced with Dex
(1 µM), or CpdA (10 µM) for 24 hr. Control wells received an equal amount of ethanol. Total RNA was
reverse transcribed and the cDNA obtained subjected to PCR analysis with primers to detect mCBG and GAPDH (housekeeping gene used as loading control), in separate reactions. PCR products were analyzed on agarose gel and visualized under UV light. The figure is representative of three independent experiments.
2
Fig. S2: Transactivation of GRE-containing promoter reporter construct. (A) Transactivation of
GRE-containing promoter by CpdA and Dex in the absence or presence of co-transfected rGRαααα (pSVGR1). BWTG3 cells (5x104 cells per well in 24-well tissue culture plates) were transiently
transfected with 360 ng GRE-containing promoter reporter construct ((GRE)250IL6PLuc), 200 ng rGRα
(pSVGR1) or pGL2-basic as indicated, and 40 ng pPGKβGopbA. Twenty-four hours after transfection,
test compounds were added (CpdA at 10 µM; Dex at 1 µM) for 24 hrs. Control wells received an equal
amount of ethanol. Luciferase values were normalized for β–galactosidase and values plotted as a
percentage of the average control. Statistical analysis was done to (i) compare values in the presence of test compounds relative to the corresponding control using one-way ANOVA followed by Dunnett’s
multiple comparison’s posttest (*P<0.05; **P<0.01) and to (ii) compare values without GR (–rGRα) to
values with co-transfected GR (+rGRα) for each compound tested using a two-tailed unpaired t-test
(aP<0.05; bP<0.01). (B) Transactivation of GRE-containing promoter by CpdA and Dex in HepG2 cells,
in the absence or presence of RU486. The (GRE)2tkLuc promoter reporter construct (360 ng) was
transiently transfected into HepG2 cells (5x104 cells per well in 24-well tissue culture plates), together
with 200 ng human GRα (pRS-hGRα) expression vector and 40 ng pPGKβGopbA. Twenty-four hours
after transfection cells were treated for 24 hrs with 10 µM test compounds as indicated. Control wells
received an equal amount of ethanol. Luciferase values were normalized for β–galactosidase and plotted
as a percentage of the average transactivation by Dex alone. Statistical analysis was done to compare values in presence of test compounds relative to the corresponding controls using one-way ANOVA followed by Dunnett’s multiple comparison’s posttest (*P<0.05; **P<0.01).
Dex (10µµµµM) CpdA (10µµµµM) RU486 (10µµµµM) 1 2 3 4 5 6 7 8 0 25 50 75 100 125 -+ + -+ -+ -+ + + + + -+ -+ + -*** *** ** * n o r m a li ze d l u c if e r a se a c ti v it y (% o f D E X t r a n sa c ti v a ti o n ±±±± S E M ; n = 4 ) 0 500 1000 1500 2000 2500 -rGRαααα +rGRαααα Dex Control CpdA ***,b *** n o r m a li ze d l u c if e r a se a c ti v it y ( % o f c o n tr o l±±±± S E M ; n = 6 ) A B
Fig. S3: Effect of pre-incubation. BWTG3 cells were incubated with 10µM CpdA and 10µM Dex for
1hr followed by washout and whole cell binding with 1nM 3H-Dex. Results show specific binding.
Statistical analysis was done to compare values in the presence of test compounds relative to the corresponding control using one-way ANOVA followed by Dunnett’s multiple comparison’s posttest (*: P<0.05; **: P<0.01). 0 500 1000 1500
Preincubation
Dex (10
µµµµ
M)
-
+
-CpdA (10
µµµµ
M)
-
-
+
S
p
e
c
if
ic
b
in
d
in
g
c
p
m
/m
g
p
r
o
te
in
±±±±
S
E
M
,
n
=
4
1nM [
3H]-Dex
4 0 25 50 75 100 M -6 CpdA 10 M -6 Dex 10 Control
Cytoplasmic Both Nuclear
% cellular distribution±±±± SEM; n=3
rGR
PI
Fig. S4: Nuclear translocation of endogenous GR in BWTG3 cells. BWTG3 cells (5 x 105 cells per
well in 6-well plates) were serum-starved in Opti-MEM for 24 h before induction with Dex (1 µM) or
CpdA (1 µM) for 30 min. Control wells received an equal amount of ethanol. After fixation, cells were
subjected to immunostaining with anti-GR, followed by anti-rabbit-Alexa 488 as a secondary Ab (GR; green). Propidium iodide staining (PI; red) was used to visualize nuclei. Cells were analysed using an Zeiss confocal LSM410 microscope. The percentage of cells showing GR in either the nuclear or cytoplasmic or both compartments was quantified for three independent experiments and is presented graphically.
Fig. S5: Transactivation of transiently transfected GRE-containing promoter reporter construct via low levels of GR, MR, PR, or AR, or ERE-containing promoter reporter construct via low levels of ER. COS-1 cells (2x106 cells per plate in 10 cm tissue culture dishes) were transiently transfected with
9 µg pTAT-GRE2-Elb-luc, and 3 ng pRS-hGRα, pRS-hMR, pSG5hPRB or pSVARo as indicated, or 9 µg
pGL2-3x-ERE-TATA-luc, and 3 ng pcDNA3-ERα. Twenty-four h after transfection cells were replated
(5x104 cells per well in 24-well tissue culture plates). Cells were induced for 24 h with solvent (ethanol)
or agonist (10-6 M) 24 h after replating). Luciferase values were normalized with protein concentration
and values plotted as fold-induction relative to average solvent. Agonists used: Dex for GR; aldosterone
for MR; R5020 for PR; DHT for AR; E2 for ER.
