Supplementary Materials and Methods
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Supplementary Materials and Methods Real-time RT-PCR

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Supplementary Materials and Methods Real-time RT-PCR

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Supplementary materials and methods real time rt pcr

Supplementary Materials and Methods

Real-time RT-PCR

Genomic DNA was isolated from washed cell pellets of eight biliary tract cancer cell lines (SNU-245, 308, 478, 869, 1079, 1196, HuCCT1, and TFK-1) and MKN45 cells using Qiagen QIAamp DNA kit (Qiagen, Hilden, Germany). The primers used in the PCR

reaction were as follows: MET, forward primer 5’-TGTTGCCAAGCTGTATTCTGTTTAC-3’ and reverse primer 5’-TCTCTGAATTAGAGCGATGTTGACA-3’; β-actin, forward primer 5’-TCATCACCATTGGCAATGAG-3’ and reverse primer 5’-CACTGTGTTGGCGTACAGGT-3’.

Amplification reactions were conducted in 20 µL volumes for 94 ℃ for 5 min, 25 cycles at 94 ℃ for 30 s, 53 ℃ for 30 s, and 72 ℃ for 30 s, followed by a final extension for 10 min at 72 ℃. The levels of MET gene mRNA were quantitatively analyzed via real-time RT-PCR assays with

SYBR Green I (Molecular Probe) using an Cycler instrument (Bio-Rad) with more than duplet reactions. Each relative mRNA expression level was calculated by normalization to the mean volume of β-actin.

Cell Growth Inhibition Assay

Tetrazolium dye (MTT; Sigma-Aldrich) assays were used to evaluate the growth inhibitory effect of PF00299804. The cells were seeded on 96-well plates, incubated for 24 h, and then treated with PF00299804 for 72 h at 37℃. After drug treatment, MTT solution was added to each well and incubated for 4h at 37℃ before the medium was removed. Then, DMSO was added and shaken for 30 min at room temperature. Cell

viability was determined by measuring absorbance at 540 nm in a microplate reader (Versa Max, Molecular Devices). Six replicate wells were

used for each analysis, and at least three independent experiments were conducted. Data points shown represent the mean while bars represent

the SE.

To evaluate the effects of PF00299804 administered in conjunction with 5-FU or cisplatin, cells were treated with serial dilutions of each drug individually and with both drugs simultaneously at a fixed ratio. Two BTC cell lines (SNU308 and SNU478) were exposed to

PF00299804 with 5-FU or cisplatin at a ratio of 1:10 (0.005, 0.01, 0.05, 0.1, 0.5 μmol/L) or 1:100 (0.001, 0.002, 0.004, 0.008,

0.016 μmol/L). The other BTC cell lines (SNU-245, 869, 1079, 1196, HuCCT-1, and TFK-1) were exposed to PF00299804 with

5-FU or cisplatin at a ratio of 1:1(0.05, 0.25, 0.5, 2.5, 5 μmol/L) or 1:10 (0.01, 0.05, 0.1, 0.5, 1 μmol/L).

After 72 h of exposure, cell viability was measured using an MTT assay. The methods described by Chou and Talalay were then used to

determine if a synergistic effect existed. Analysis of the median effect was conducted using the Calcusyn software (Biosoft) to

determine a combination index value (CI > 1, antagonistic effect; CI = 1, additive effect; CI <1, synergistic effect).

Western Blot Analysis

Cells were incubated with PF00299804 in 10% FBS media. After 24 h, the cells were treated with lysis buffer. The same amount of protein was

then obtained from each suspension and subjected to SDS-PAGE, after which it was transferred to nitrocellulose membranes. After blocking with buffer, the membrane was incubated with primary antibodies at 4℃ overnight. Antibodies against RRM1 were purchased from Abcam

(Cambridge, UK), and TS, and MET antibodies were obtained from Cell Signaling Technology (Beverley, MA, USA), and anti-actin antibody

was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA).


Supplementary materials and methods real time rt pcr

Supplementary figure 1

SNU 478

SNU 869

PF00299804

PF00299804

C

C

RRM1

Actin

Supplementary Figure 1. RRM1 downregulation by PF00299804 treatment in SNU478 cells. SNU478 and SNU869 cell

lines were treated with increasing doses of PF00299804 (0.01, 0.1, 1 μmol/L) for 24h, after which the extracts were

immunoblotted with RRM1 antibodies.


Supplementary materials and methods real time rt pcr

Supplementary figure 2

MKN45 (MET amp)

SNU1079

SNU1196

HuCCT-1

TFK-1

SNU245

SNU308

SNU478

SNU869

MET

Actin

Fold MET amplification

Supplementary Figure 2. Fold MET amplification in BTC cell lines. Protein expression level of MET was analyzed by immunoblotting in

BTC cell lines and MET amplified MKN45 gastric cancer cell line (positive control; upper). Fold MET amplification was analyzed in genomic DNA isolated from BTC cell lines and MKN45 cells by real-time PCR(bottom).


Supplementary materials and methods real time rt pcr

Supplementary figure 3

SNU 308

SNU 478

SNU1079

PF00299804

PF00299804

PF00299804

C

C

C

TS

Actin

Supplementary Figure 3. TS downregulation by PF00299804 treatment. SNU308, SNU478 and SNU1079 cell lines were treated with

increasing doses of PF00299804 (0.01, 0.1, 1 μmol/L) for 24h, after which the extracts were immunoblotted with TS antibodies.


Supplementary materials and methods real time rt pcr

Supplementary Table 1. Combination of PF00299804 with 5-FU in biliary tract cancer cell lines

NOTE: Shown are the IC50 values of PF00299804 or 5-FU using MTT assay. Combination index for the combination of

PF00299804 with 5-FU at the 50% fraction affected was calculated by the Chou and Talalay method in biliary tract cancer cell

lines (CI > 1: antagonistic effect, CI = 1: additive effect, CI <1: synergistic effect).


Supplementary materials and methods real time rt pcr

Supplementary Table 2. Combination of PF00299804 with cisplatinin biliary tract cancer cell lines

NOTE: Shown are the IC50 values of PF00299804 or cisplatin using MTT assay. Combination index for the combination of

PF00299804 with cisplatin at the 50% fraction affected was calculated by the Chou and Talalay method in biliary tract cancer cell

lines (CI > 1: antagonistic effect, CI = 1: additive effect, CI <1: synergistic effect).


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