Knockdown of Rad9A enhanced DNA damage induced by Trichostatin A
in esophageal cancer cells
Xueli Pang1, Gang He2, Chao Luo2, Yan Wang2, Bo Zhang2
Supplementary materials
Contents:
Table S1 Primers for Realtime PCR
Table S2 Sequences of siRNA against Rad9A and negative control (NC)
Figure S1 DNA damages induced by TSA in KYSE150 cells.
Figure S2 DNA damages induced by TSA in EC109 cells were evaluated by single-cell
gel electrophoresis.
Figure S3 Gene expression of Rad9A in KYSE150 cells.
Figure S4 DNA damages induced by TSA in EC109 cells treated with SiRNA against
Rad9A were evaluated by single-cell gel electrophoresis.
Table S1 Primers for Realtime PCR
Target Name
Target sequence
Products(bp)
F: TCTCAGACACCGACTCGCACTC
Rad9A mRNA
194
R: AGGTGAAAGGGAAATGGAGGG
F: GCCTGGGTGACAGAGCGAGACT
Rad9A P1*
125
R: ACCTCGGGCACTGGCATCCTT
F: AGGGTCCTCAAGGTCATAGAA
Rad9A U1*
124
R: GAATAAGGGTAGATGCCGAAG
F: GGGAAGGTGAAGGTCGGAGTC
GAPDH mRNA
232
R: CCTGGAAGATGGTGATGGGAT
F: GAGGGACTGGGGGAGGAGGGAA
P21
267
R: CCACAAGGAACTGACTTCGGCA
F: GATTTCGTTGGAAACGGGATG
α-Satellite
102
R: AAGGGAATGTTCAATTCTGTG
* P1 indicated the promoter region of Rad9A, and U1 indicated upstream region of Rad9A
which located about 5Kb from the transcription start site.
Table S2 Sequences of siRNA against Rad9A and negative control (NC)
Target Name
Target sequence
5’ GUCUUUCCUGUCUGUCUUCTT
Rad9A SiRNA
5’ GAAGACAGACAGGAAAGACTT
5’ UUCUCCGAACGUGUCACGUTT
NC SiRNA
5’ ACGUGACACGUUCGGAGAATT
Figure S1
Figure S1: DNA damages induced by TSA in KYSE150 cells. (A) KYSE150 cells
were treated with increasing concentration of TSA (0~1.0μM) for 24h and then total
proteins were harvested for Western blotting analysis. (B) KYSE150 cells were
treated with TSA (0.2μM) for indicated periods and total proteins were harvested for
Western blotting analysis. Significantly increased protein level of H2Ax induced by
TSA treatment was observed in KYSE150 cells.
A
B
Figure S2 EC109 cells were treated with or without TSA (0.2μM) and harvested for the
single-cell gel electrophoresis. Cells were analyzed with a fluorescence microscope.
The frequency of comets in 200 nuclei served as indicators. Data were the mean ± SD
of three independent experiments. *P< 0.01 by t-test.
Figure S3 Gene expression of Rad9A in KYSE150 cells. After KYSE150 cells were
treated with increasing concentration of TSA for 24h, total proteins were harvested for
Western blotting analysis. No obviously induction of Rad9A by TSA was detected in
KYSE150 cells.
Figure S4 EC109 cells were transfected with siRNA against Rad9A or a negative
control (NC). Twenty-four later, cells were further treated with or without TSA (0.2μM)
for 24h. Then cells were harvested for single-cell gel electrophoresis. Cells were
analyzed with a fluorescence microscope. The frequency of comets in 200 nuclei
served as indicators. Data were the mean ± SD of three independent experiments.
*P< 0.01 by t-test.