ONLINE EXPANDED EXPERIMENTAL PROCEDURES
mRNA and protein expression analysis. Total RNA was isolated using
Trizol reagents (Invitrogen), reverse transcribed, and analysed by quantitative
PCR (qPCR) using SYBR Green and the Mastercycler ep realplex2 system
(Eppendorf, Hamburg, Germany). Primers for 36B4 were included for
normalization. For semi-quantitative RT-PCR, cDNA was amplified by using
the following parameters: 94℃ for 30 s, 57℃ for 30 s and 72℃ for 30 s, with a
final extension step at 72℃ for 7 min. PCR products were electrophoresed
through 2% agarose gel, stained with ethidium bromide and visualized under
ultraviolet illumination. A complete list of PCR primers is shown in
Supplementary Table 1. For protein analysis, we directly lysed cells in RIPA’s
buffer. The protein concentration was quantified with a Dc protein assay
reagent (Bio-Rad, Hercules, USA). Equal amount of protein was loaded and
separated by 10% SDS-PAGE and then transferred onto polyvinylidene
difluoride membrane (Bio-Rad). The membranes were incubated overnight
with appropriate primary antibodies. Bound antibodies were then visualized
using alkaline phosphatase-conjugated secondary antibodies. Quantitative
analysis was performed by NIH Image J 1.32j software. For antibody
information, anti-BAF60a, anti-RORα, anti-Brg-1, anti-Ini1, and anti-BAF155
antibodies were purchased from Santa Cruz Biotech, anti-Brm antibody was
obtained from Abcam, and anti-β-Actin and anti-Flag antibodies were from
Sigma.
Reporter gene assays. Reporter gene assays were performed in HepG2
cells. In a typical experiment, 50 ng of reporter plasmids were mixed with 20 ng
of expression constructs for transcription factors in the presence or absence of
BAF60a expression construct. Equal amounts of DNA were used for all
transfection combinations by adding appropriate vector DNA. Relative
luciferase activities were determined 48 h following transfection. All
transfection experiments were performed in triplicates.
Glucose production assay. Mouse primary hepatocytes were isolated
using traditional two-step collagenase digestion method and infected with
adenoviruses expressing GFP or BAF60a for 2 days before incubation for 6 h
in 1 ml of glucose-free DMEM medium (pH 7.4), without phenol red,
supplemented with 20 mM sodium lactate and 2 mM pyruvate. The medium
was assayed for glucose with a colorimetric kit (Sigma). Values were
normalized to the protein content of the whole-cell lysates.
ChIP assay. Chromatin immunoprecipitation was performed essentially
as described by the Upstate Biotechnology. Briefly, HepG2 cells were
transduced with green fluorescent protein (GFP) or BAF60a adenoviruses for
48 h. Chromatin lysates were prepared, pre-cleared with Protein-A/G agarose
beads, and immunoprecipitated with antibodies against BAF60a (Santa Cruz
Biotech), K9-dimethylated and K4-trimethylated histone H3 (Abcam),
acetylated histone H3 (Upstate Biotechnology), or normal mouse IgG (Santa
Cruz Biotech) in the presence of BSA and salmon sperm DNA. Beads were
extensively washed before reverse cross-linking. DNA was purified using a
PCR purification kit (Qiagen) and subsequently analysed by PCR using
primers flanking the proximal RORE on the human Bmal1 and G6Pase
promoter, as detailed in Supplementary Table 1.
Metabolic parameters. Food was removed at ZT0, and mouse blood
samples were collected at ZT4 in order to obtain postprandial values. Blood
samples were collected from vena cava and the plasma was isolated and
stored at -80℃ until analysis. Glucose levels were determined using an
Accu-Check glucometer (Roche Diagnostics). Triglyceride and free fatty acid
concentrations were determined by enzymatic assays with the use of
commercial kits according to the manufacturer’s protocols. The plasma insulin
concentration was measured using an enzyme-linked immunosorbent assay
(ELISA) kit from Linco Research.
Figure Legends
Fig. S1. Diurnal expression of SWI/SNF complex subunits in mouse liver.
qRT-PCR analysis of indicated subunit mRNA expression in the liver from
wild-type mice entrained to an LD 12:12 cycle. Data are represented as mean
± s.d. *P<0.04, peak versus nadir.
Fig. S2. The expression pattern of PGC-1α, ApoB and FAS is not altered in the
liver with BAF60a knockdown. qRT-PCR analysis of PGC-1α, ApoB and FAS
mRNA expression in pooled liver from mice injected with BAF60a shRNA or
control adenoviruses via tail vein. Shown with mean ± s.d.
Fig. S3. RORγ does not mediate the stimulatory effect of BAF60a on Bmal1
transcription. Reporter gene assays were performed using Bmal1-luciferase
reporters. Data are represented as mean ± s.d.
Fig. S4. Comparison of mRNA abundance of BAF60 family members in HepG2
and HEK293 cells. Semi-quantitative RT-PCR analysis of BAF60a, BAF60b,
and BAF60c mRNA expression in HepG2 (Panel A) and HEK293 (Panel B)
cells.
Fig. S5. qPCR analysis of ChIP assays with the indicated antibodies using
HepG2 cells transduced with GFP or BAF60a adenoviruses. PCR primers
amplify a fragment flanking the proximal RORE on the Bmal1 (Panel A) or
G6Pase promoter (Panel B). Data are represented as mean ± s.d. *P<0.05,
BAF60a (open box) versus GFP (filled box).