SIRT6 promotes the COX-2 pathway and acts as an oncogene in
skin cancer
Mei Ming1, Weinong Han1, Baozhong Zhao1, Nagalingam R. Sundaresan2,3, Chu-Xia
Deng4, Mahesh Gupta2, and Yu-Ying He1*
Supplemental information: Materials and Methods
Determination of UVB-induced CPD/6-4PP level in genomic DNA by slot blot assay
Slot blot assays of CPD/6-4PP were performed as described previously (36). Briefly,
cells were collected at different time points post-UVB, and DNA was isolated using a
QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). The DNA concentration was
calculated from the absorbance at 260 nm using NanoDrop 1000 (NanoDrop products,
Wilmington, DE, USA). The CPD in DNA were quantified by slot-blot (Bio-Rad, Hercules,
CA, USA) with a monoantibody (TDM-2 for CPD and 6-4PP, COSMO BIO Co., Tokyo,
Japan) as described previously (37). The chemiluminescence was detected with a
Carestream Imaging Station (Carestream, Rochester, NY, USA). For examining repair
kinetics, the percentage of repair was calculated by comparing the optical density at the
indicated time with that of the corresponding absorbance at time zero, when there was
no opportunity for repair, and 100% of CPDs were present post-UVB.
Ming et al Supplemental Figure S1
Supplemental Figure S1
A
B
siSIRT6 -
Hours post-UVB
0
6
24
NC
CPD
siSIRT6
+
SIRT6
NC
siSIRT6
GAPDH
6-4PP
E
NC
UVB - + +
Hours 1.5 6
SIRT6
siSIRT6
- + +
1.5 6
p-Chk1
Chk1
p-Chk2
Chk2
D
C
Ad-SIRT6
SIRT6
GAPDH
-
+
Hours post-UVB
0
6
24
CPD
EV
Ad-SIRT6
EV
Ad-SIRT6
ac-p53
p53
p21
6-4PP
gH2AX
GAPDH
Figure S1. SIRT6 has no effect on UVB-induced DNA damage repair and
checkpoint activation. A, Immunoblot analysis of SIRT6 and GAPDH in NHEK cells
transfected with NC or siSIRT6. B, Slot-blot analysis of CPD and 6-4PP in NHEK cells
transfected with NC or siSIRT6 at 0, 6, or 24 h post-UVB irradiation. C, Immunoblot
analysis of SIRT6 and GAPDH in NHEK cells infected with an adenoviral vector
expressing empty vector or SIRT6 (Ad-SIRT6). D, Slot-blot analysis of CPD and 6-4PP
in NHEK cells infected with an adenoviral vector expressing empty vector or SIRT6 (AdSIRT6) at 0, 6, or 24 h post-UVB irradiation. E, Immunoblot analysis of SIRT6, p-Chk1,
Chk1, p-Chk2, Chk2, ac-p53, p53, p21, H2AX, and GAPDH in NHEK cells transfected
with NC or siSIRT6 at 1.5 or 6 h post-UVB or -sham irradiation.