Supplementary information
Supplementary Figure S1
(a) REV-ERB protein levels in breast cancer BT-474, MCF-7, SK-BR-3, MDA-MB-361 and normal human
epithelial HMEC cell lines were evaluated by immunoblot analysis with an -REV-ERB specific antibody.
Densitometry analysis of protein signals is reported as relative protein levels normalized by GAPDH. Value if
REV-ERB signal from HMEC sample was set to 1. Shown as mean ± SEM, n=3.

P < 0.001, cancer
versus normal cells. (b) The amounts of the ERBB2, REV-ERB and the reference GAPDH genes were
measured in ERBB2-amplified breast cancer BT-474 and normal human epithelial HMEC cells by qPCR.
Representative ERBB2, REV-ERB and GAPDH amplification curves obtained from 2000 genome
equivalents (assuming 3 ng of genomic DNA as 1000 haploid genome-equivalents1). (c) Relative copy
number (Q) of ERBB2 vs GAPDH and REV-ERB vs GAPDH in BT-474 and HMEC cells was calculated as
described previously1. Indicating a similar copy number of REV-ERB among the two cell lines, Q value for
REV-ERB vs GAPDH in BT-474 and HMEC cells was within the cut-off limits reported for amplifications and
deletions (1.3 and 0.7, respectively)1.
Supplementary Figure S2
Relative abundance of REV-ERB and REV-ERB transcripts in the ORIGENE (USA) Healthy tissues cDNA
collection, which contains cDNA of various tissues pooled from multiple healthy individuals of different
ethnicity to avoid detection of individual differences. Presented as mean of the percentage of each isoform
expression contribution to the total REV-ERBs expression.
Supplementary Figure S3
Relative abundance of REV-ERB and REV-ERB transcripts analyzed from the ORIGENE (USA) colon (a),
liver (b), and prostate (c) cancer tissues cDNA collection. Presented as mean of the percentage of each
isoform expression contribution to the total REV-ERBs expression. Dash line refers to the REV-ERB
percentage in normal tissue.
Supplementary Figure S4
(a) MCF-7 cells were transfected with vectors co-expressing a GFP protein with shRNA sequences against a
non-targeting (Control), REV-ERB (shREV-ERB), REV-ERB (shREV-ERB) genes. Forty eight hours
post-transfection, GFP-positive cells were sorted by FACS and processed for qRT-PCR analysis to evaluate
the expression of REV-ERB-regulated genes. Relative expression was determined using GAPDH for
normalization. HRPT expression is reported as representative of a REV-ERB-independent gene. Control
value was set to 1. Data are shown as mean ± SEM, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001, shRNA
versus control. (b) HEK-293 cells were transfected and analyzed as in (a). Data are shown as mean ± SEM,
n = 3. ** P < 0.01 and *** P < 0.001, shRNA versus control.
Supplementary Figure S5
The expression of REV-ERB-regulated BMAL1 and PEPCK genes was analyzed in HMEC cells 72 h after
transfection with pooled siRNA sequences against REV-ERB (siREV-ERB), REV-ERB (siREV-ERB)
and both REV-ERB and REV-ERB (siREV-ERBs), with a non-targeting pool as a negative control
(Control). Relative expression was determined by qRT-PCR using GAPDH for normalization. HRPT
expression is reported as representative of a REV-ERB-independent gene. The effect of REV-ERB silencing
on the two nuclear receptor variants was also evaluated. Shown as mean ± SEM, n = 3. *P < 0.05 and ***P <
0.001, siRNA target sequences versus control. #P < 0.05 siREV-ERBs versus REV-ERB or REV-ERB
Supplementary Figure S6
(a) BT-474 cells were transfected with pooled siRNA sequences against REV-ERB (siREV-ERB), REVERB (siREV-ERB), and with a non-targeting pool as a negative control. Relative siRNA target transcript
levels at the indicated post-transfection times was determined by qRT-PCR setting the value in control
sample as 1. Data are shown as mean ± SEM, n = 3. (b) BT-474 cells were transfected with vectors coexpressing a GFP protein with shRNA sequences against a non-targeting (Control), REV-ERB (shREVERB), REV-ERB (shREV-ERB) genes. Green cells were counted at intervals and expressed as
percentage of control as previously described2.
Supplementary Figure S7
Co-transfection assay in HEK-293 cells with REV-ERB (a) or REV-ERB (b), and a luciferase reporter
driven by two repeats of a RevRE consensus illustrating the antagonistic activity of SR8278 toward the two
REV-ERB analogs. Data expressed as fold increase of luciferase activity versus vehicle. EC50 for SR8278
antagonism versus REV-ERB and REV-ERB is indicated. Data expressed as mean ± SEM, n = 6.
Supplementary Figure S8
(a) BT-474 cells were transfected with pooled siRNA sequences against ATG5 gene and with a nontargeting pool as a negative control (Control). After 72 h, ATG5 expression was determined by qRT-PCR
using GAPDH for normalization. ATG5 expression in Control sample was set to 1. Data are shown as mean
± SEM, n = 3. P < 0.001, siATG5 versus Control. (b) Representative immunoblot analysis to validate ATG5
protein knock-down by siRNA interference as in (a). GAPDH was used as loading control. (c) Twelve hours
after transfection with siRNA sequences, ATG5-silenced and control cells were treated 48 h with vehicle, 10
M SR8278 or 2.5 M vorinostat. The percentage of cells with a caspase-3 and -7 activity induction (%
caspase-positive cells) was evaluated with the Image-iT LIVE Red Caspase-3 and -7 Detection Kit
(Invitrogen). P < 0.05 and P < 0.01, compound-treated siATG5 versus compound-treated control cells.
Supplementary Figure S9
(a) Immunoblot analysis with the indicated antibodies of protein samples from BT-474 cells treated 24 h with
vehicle or two concentrations of the REV-ERB antagonist SR8278 (3 and 10 M). Densitometry analysis of
protein signals is reported as relative protein levels normalized by GAPDH. Vehicle sample value was set to
1. Shown as mean ± SEM, n=3. (b) BT-474 cells were transfected with pooled siRNA sequences against
REV-ERB (siREV-ERB) or a non-targeting pool as a negative control (Control) and then treated 2 h with
vehicle (water) or 25 M Chloroquine (CQ). The levels of LC3, p62, and GAPDH proteins were analyzed by
immunoblot analysis with specific antibodies. Densitometry analysis of protein signals is reported as relative
protein levels normalized by GAPDH. Vehicle-treated Control sample value was set to 1. Shown as mean ±
SEM, n=3. P < 0.05, CQ versus vehicle.
Supplementary Figure S10
(a) Purification of the REV-ERB Ligand Binding Domain (LBD) for
19F-NMR-based
screening. Left, REV-
ERB LBD fused with a Maltose Binding Protein (MBP) was induced for 4 h with 0.3mM IPTG in E.coli BL21
cells. After extraction of the soluble protein fraction (Soluble lysate) the MBP-REV-ERB LBD was purified by
affinity chromatography with amylose-agarose beads and eluted with 10mM Maltose. A representative gel of
the purification steps stained by Coomassie is reported. (b) After digestion over-night with the Factor Xa
protease, REV-ERB LBD and MBP products were separated by affinity chromatography with a
hydroxyapatite column: both products were bound to the column at pH 5.8 in 50 mM sodium phosphate;
150mM NaCl and REV LBD was then eluted at pH 6.6 in 50 mM sodium phosphate; 150 mM NaCl. The MBP
product was further eluted at pH 7.2 with high salt condition (50 mM sodium phosphate; 500 mM NaCl). A
representative gel of the eluted product stained by Coomassie is shown. (c) For
19F-NMR
experiments
different mixtures containing 4/5 fluorinated compounds were tested in presence or absence of 2 M
recombinant REV-ERB LBD. A representative spectra of the mixture containing ARN5187 (1) is reported,
showing a specific line broadening effect of ARN5187 (1)
19F-NMR
signal. (d) Cytotoxicity of 100M
ARN5187 as evaluated in BT-474 and HEK-293 at the indicated post-treatment time points. Reported as
percentage number of cells versus Vehicle-treated samples. Shown as mean ± SEM, n = 6.
Supplementary Figure S11
Co-transfection assay in HEK-293 cells with REV-ERB and a luciferase reporter driven by two repeats of a
RevRE consensus illustrating the antagonistic activity of ARN5187 toward REV-ERB isoform. Data
expressed as fold increase of luciferase activity versus vehicle (DMSO-d6). EC50 for ARN5187 antagonism
versus REV-ERB was 16 ± 3.8 M. Data expressed as mean ± SEM, n = 6.
Supplementary Figure S12
Immunoblot analysis with the indicated antibodies of protein samples from BT-474 cells treated 2 or 4 h with
vehicle, 25 M ARN5187 and 25 M chloroquine (CQ). Densitometry analysis of protein signals is reported
as relative protein levels normalized by GAPDH. Vehicle sample value was set to 1. Shown as mean ± SEM,
n = 3. P < 0.05 and P < 0.01, compounds versus vehicle.
Supplementary Figure S13
(a) Low magnification of a BT-474 cell exposed for 2 h to ARN5187. The cytoplasm contains numerous AVds
of different sizes (white asterisks). Inset: high magnification of a single AVd. Note the electron-dense
materials (arrowheads). (b) BT-474 cell exposed for 2 h to CQ. Numerous AVds are present inside the cell
cytoplasm (white asterisks). Inset: high magnification of two AVds containing electron dense material
(arrowheads). Abbreviations: m, mitochondria, n, nucleus. Scale bars are 2 µm for (a) and (b); 1 µm insets.
Supplementary Figure S14
(a) Representative UPLC/MS spectra of ARN5187. (b) Representative 1H-NMR spectra of ARN5187.
Supplementary Figure S15
Representative HSQC (upper part) and HMBC (lower part) spectra of ARN5187 showing the structural
identity of the synthesized product.
Supplementary Figure S16
(a) Representative immunoblot analysis to validate REV-ERB protein knock-down by siRNA interference in
BT-474 cells. Because none of the tested anti-REV-ERB antibodies (see Supplementary Table S2) was
able to recognize the endogenous REV-ERB protein, we tested by immunblot analysis with an anti-Flag
antibody the reduction of a Flag-tagged REV-ERB co-transfected in BT-474 cells with pool siRNA
sequences against REV-ERB (siREV-ERB) or non-targeting sequences (Control). A GFP protein coexpressed with the Flag-tagged REV-ERB product was used for normalization samples for transfection
efficiency. (b) Representative immunoblot analysis to validate REV-ERB protein knock-down by siRNA
interference in BT-474 cells transfected with pool siRNA sequences against REV-ERB (siREV-ERB) or
non-targeting sequences (Control). GAPDH was used as loading control. (c) HEK-293 cells were cotransfected with a Flag-REV-ERB expressing vector and a plasmid co-expressing a GFP with either a
shRNA sequence against REV-ERB (shREV-ERB) or a non-targeting sequence (control). After 48 h, the
levels of Flag-REV-ERB was analyzed by immunoblot analysis with an anti-Flag antibody and normalized
using the GFP signal as transfection control. (d) HEK-293 cells were transfected with a plasmid coexpressing a GFP with either a shRNA sequence against REV-ERB (shREV-ERB) or a non-targeting
sequence (Control). After 48 h, GFP-expressing cells were sorted by FACS and processed for immunoblot
analysis with an anti-REV-ERB antibody. GAPDH was used as loading control.
Supplementary Table S1
Primer sequences used in the present study.
Supplementary Table S2
Antibodies and immunoblot dilution used in the present study.
Supplementary Table S3
REV-ERB and REV-ERB iRNA sequences used in the present study.
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