Silencing expression of ribosomal protein L26 and L29 by RNA
interfering inhibits proliferation of human pancreatic cancer
PANC-1 cells
Chaodong Li 1, 2, Mei Ge 2, 3, Yu Yin 2, 3, Minyu Luo 2, Daijie Chen 1, *
1 School of Biotechnology, East China University of Science and Technology, Shanghai, 200237,
People’s Republic of China
2 Shanghai Laiyi Center for Biopharmaceutical R&D, Shanghai, 200240, People’s Republic of China
3 Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China
﹡Correspondence author: Daijie Chen. Address: School of Biotechnology, East China University of
Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic of China. Tel:
862134207020. Fax: 862134207023. Email address: hccbred@gmail.com.
Figure legend
Fig. S1 Silencing of RPL26 and RPL29 induces cell arrest at G0/G1 phase and enhances cell apoptosis in
P-M and P-W cells. (a) P-M cells were transfected with RPL26-siRNA or RPL29-siRNA (40 nM) for 72
h, and then cell cycle measured using FCM analysis. Compared with control (untransfected) and NC
(Mock-siRNA) groups, RPL26-siRNA and RPL29-siRNA induced P-M cells arrest at G0/G1 phase. (b)
P-M cells were treated with RPL26-siRNA or RPL29-siRNA analyzed by Annexin V-APC/7-AAD
staining using FCM analysis. The lower right area shows early apoptotic cells. Cell apoptosis was
increased compared with control (untransfected) and NC (Mock-siRNA) groups. (c) P-W cells were
transfected with RPL26-siRNA or RPL29-siRNA (40 nM) for 72, and the cell cycle measured using FCM
analysis. (d) P-W cells were treated with RPL26-siRNA or RPL29-siRNA analyzed by Annexin
V-APC/7-AAD staining using FCM analysis. Three separate experiments were of similar results.
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Materials and methods
P-M and P-W cells was maintained in vitro in DMEM high glucose medium (Gibco, CA, USA)
supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco). Cells were incubated at 37 ℃ in a
humidified incubator with 5% CO2.
P-M and P-W cells (1.5×105) were seeded in six-well plates respectively and transfected with
Mock-siRNA, RPL26-siRNA and RPL29-siRNA (40 nM, 72 h). For cell cycle analysis, the cell DNA
was stained with PI using Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China) according
to the manufacturer’s guidelines. In briefly, cells were harvested by trypsinization and fixed with cold
75% ethanol at 4 ℃ overnight. The fixed cells were collected and suspended in PBS containing 10µg/ml
PI and 10µg/ml RNase A, and then incubated at room temperature for 30 min. DNA content was analyzed
by the BD FACSCalibur (BD Biosciences), and each histogram was constructed with the data from at
10,000 events. The data were analyzed and expressed as percentages of total gated cells using the Modfit
LT™ Software (BD Biosciences). For apoptosis analysis, the cells were washed twice with ice-cold PBS
and stained with Annexin V-APC and 7-AAD by Apoptosis Detection Kit (Keygen, Nanjing, China) in
the dark at room temperature for 10 min. Then cells were analyzed with the BD FACSCalibur and FlowJo
software.
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