Study of cell biological functions of PCDH10

3.1.1 Cell culture

Human CRC cell line HCT116 (gift from Dr. SL Yu), was cultured in RPMI-1640 (GIBCO-Invitrogen) supplemented with 10% fetal bovine serum (FBS, HyClone) without antibiotics under 95% air and 5% carbon dioxide (CO2) at 37qC. Human CRC cell lines SW480 and HT29 (purchased from the Food Industry Research and Development Institute and gift from Dr. SL Yu, respectively), were cultured in DMEM (GIBCO-Invitrogen) supplemented with 10% FBS (HyClone) without antibiotics under 95% air and 5% CO2 at 37qC.

3.1.2 Vector construction

Vector construction was done by previous senior coworkers Yen-Chun Lai and Chao-Ha Fu. Briefly, PCDH10 V1 and PCDH10 V2 cDNA was obtained from MCF7 and were cloned into the mammalian expression vector pcDNA3.1/V5-His-TOPO (Invitrogen, Appendix Figure 1). The two constructs were named as pcDNA3.1/PCDH10.v1 and pcDNA3.1/PCDH10v.2 for PCDH10 V1 and PCDH10 V2, respectively.

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3.1.3 Cell transfection and stable clone selection

Transfection was performed using Lipofectamine^TM 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 18h before transfection, 5x10⁵ cells per well of a 6-well plate were seeded (~90% confluence) in 2mL RPMI-1640 containing 10% FBS.

During transfection, Lipofectamine^TM 2000 and plasmid DNA were mixed in a 2.5:1 ratio (10μL Lipofectamine^TM 2000 and 4μg plasmid DNA purified by QIAGEN Plasmid Midi Kit). Both were diluted with 250μL of Opti-MEM Ι Reduced Serum Medium (Invitrogen) before complexing and allowed to stand at room temperature for 5 min. The diluted Lipofectamine was then mixed with the diluted plasmid DNA and incubated at room temperature for 20 min. The complexes were then added to the cells and incubated at 37qC in a 5% CO2 incubator for 6 h. Medium will be changed back to RPMI-1640 with 10% FBS only and incubated at the same condition for a further 18h (Total 24h since the time of introduction of the complexes). Part of the cells will then be collected for western blotting to confirm protein expression. For stable clone selection, 1200μg/mL G418 (Geneticin, GIBCO) was added 24h after transfection and kept selecting for at least 2-3 weeks. Single stable clones obtained were then kept at a final concentration of 600μg/mL G418.

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3.1.4 Western blotting

Total cellular extracts were obtained by RIPA lysis buffer with protease inhibitor.

Protein concentrations were determined by the Bradford method (Bio-Rad). 4μL 4x sodium dodecyl sulfate loading dye was added to 12μL sample, and the sample was boiled for 10 min. Protein samples (50μg per lane) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was then blocked with 5% skim milk in Tris-HCl buffered saline containing 0.025% Tween-20 (TBST) at room temperature for 1h followed by incubation with the primary antibody for 1.5h at room temperature or overnight at 4qC. The membrane was then washed with TBST thrice (10 min per wash) and then incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1h at room temperature. The membrane was then washed as previously described. The bands were visualized using the Western Lightning Chemiluminescence Reagent Plus (PerKinElemer) and detected by Fujifilm LAS 4000 Imager.

3.1.5 Cell proliferation assay

MTT assay for cell proliferation was performed for single stable clones of PCDH10

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V1 and PCDH10 V2. Briefly, 3000 cells per well were seeded in a 96-well plate in four replicates. The colorimetric MTT (Amresco) assay was used to measure cell numbers at 24, 48, 72, 96 and 120h. 50μg MTT was added to each well and incubated for 90 min at 37qC, followed by dissolving the formazan by 100μL Dimethyl sulfoxide (DMSO). Absorbance at 570nm was then measured.

3.1.6 Camptothecin treatment

Camptothecin was isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree). It binds irreversibly to the DNA-topoisomerase I complex, and traps the enzyme in a covalent linkage with DNA, thus depleting cellular topoisomerase I. However, due to its low solubility and adverse drug effect, it is no longer adapted in chemotherapy, though derivatives of it are developed and approved for use in chemotherapy such as irinotecan.³¹ Camptothecin (Sigma-Aldrich) is employed in our study to induce apoptosis in CRC cells.

3.1.7 Flow cytometry analysis for apoptosis

1 x 10⁶ cells per well were seeded in a 6-well plate or 1.5 x 10⁵ cells per well were seeded in a 12-well plate. For medium only condition, cells were incubated in RPMI-1640 with 10% FBS with or without 600μg/mL G418 for 48h. For camptothecin treatment, cells were first plated for 24h, then 10ng/mL camptothecin (Sigma-Aldrich)

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will then be added in the medium and incubated for a further 18h. Cells treated with 0.01% DMSO at the same time interval act as the control group since camptothecin has to be dissolved in DMSO. Cells will then be collected and detected for apoptosis using ANNEXIN V-FITC Kit (Beckman Coulter) as instructed by the manufacturers. Briefly, cells were washed by cold PBS (pH7.4) twice, followed by addition of 100μL cold 1x binding buffer, and stained with 1μL of Annexin V-FITC and 5μL propidium iodide (PI). After incubation for 15 min in the dark on ice, the cells are ready to be analyzed by flow cytometry.

3.1.8 Study of epithelial-to-mesenchymal transition

TGF-β1 (PeproTech) was used to induce microsatellite stable (MSS) CRC cell lines SW480 and HT29 to undergo epithelial-to-mesenchymal transition (EMT). ³² Briefly, 5, 10 or 20ng/mL TGF-β1 was used to treat the above CRC cell lines for 24 or 48 hours with 1% or 10% FBS in order to test the optimal condition for TGF-β1 treatment.

Afterwards, cells were collected for investigation of the EMT markers, E-cadherin and vimentin by western blotting (Mouse anti-E-cadherin antibody, BD Transduction Laboratories^TM and mouse anti-vimentin monoclonal antibody, Chemicon). For the investigation of involvement of PCDH10 in EMT pathway, cells were seeded in a 6-well plate to achieve 90% confluence for each cell line used for 18h, then either 4μg

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pcDNA3.1/V5-His-TOPO or PCDH10 V1 plasmid was transfected to the cells. 24h after transfection (procedure as described above), 10ng/mL TGF-β1 was added to each well and incubated for a further 24h or 48h. Cells were then collected for investigation of the above EMT markers by western blotting.