TABLES
Supplementary Table 1: FORMATS OF CURRENT AND PROPOSED NOVEL CELL MIGRATION METHOD. A collection of existing conventional cell migration representation formats and their computation formula provides an understanding into the differences currently encountered in making effective comparison of scientific data on cell migration. Further, little attention has been paid on positive identification of silenced genes in cells under such studies. As such, a novel cell migration index has been proposed to incorporate these considerations. Legend as used in cell migration index formula column: bm = before migration, c = knockdown control (i.e. shLuc) , n = dapi-stained cells, g = gene of interest, L = at lower membrane surface or bottom of migration well, r = DsRed Express red fluorescent protein-stained cells, T = total cells on upper and lower surfaces of membrane.
s/n / Format Description / Cell Migration Index Formula / Recent References1 / Absolute cell count of migrated cells at lower membrane / / 9, 21
2 / Relative cell count of migrated cells as compared with control / / 19,22,23
3 / Relative cell count of migrated cells to total seeded cells as compared with control / / 18,20,24
4 / Normalized relative cell count of migrated cells to total seeded cells as compared with control and corrected for cell proliferative effects / / 14,15
5 / Normalized relative infected cell count (N-RICC) of migrated infected cells to ratio of seeded infected cells to total cells as compared with control and corrected for cell proliferative effects / / N-RICC
Footnotes:
21. Ketolainen JM, Alarmo EL, Tuominen VJ, Kallioniemi A (2010) Parallel inhibition of cell growth and induction of cell migration and invasion in breast cancer cells by bone morphogenetic pro- tein 4. Breast Cancer Res Treat 124(2):377–386
22.Takeda I, Maruya S, Shirasaki T, Mizukami H, Takahata T, et al. (2007) Simvastatin inactivates beta1-integrin and extracellular signal-related kinase signaling and inhibits cell proliferation in head and neck squamous cell carcinoma cells. Cancer Sci 98: 890-899.
23.Li Q, Wu M, Wang H, Xu G, Zhu T, et al. (2008) Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett 261: 55-63.
24.Backhus LM, Petasis NA, Uddin J, Schönthal AH, Bart RD, et al. (2005) Dimethyl celecoxib as a novel non-cyclooxygenase 2 therapy in the treatment of non-small cell lung cancer. J Thorac Cardiovasc Surg 130: 1406-1412.
Supplementary Table2: LIST OF TARGET AND shRNA SEQUENCES FOR CELL MIGRATION (with hairpin of “TTCAAGAGA” or “CTCGAG”, MluI or NotI as restriction site for sequencing at 3’ end, 6 T’s as Pol III terminator, BamHI overhang at 5’ end, and EcoRI overhang at 3’ end)
Gene ID / NCBI ID / TARGET SEQUENCE; shRNA SEQUENCE (5’ to 3’)ITGA5 _ #1 / NM_002205.2 / Target:CTCCACAGATAACTTCACCCGAA
shRNA: GATCCGCCACAGATAACTTCACCCGAACTCGAGTTCGGGTGAAGTTATCTGTGG TTTTTTGCGGCCGCG
ITGA5 _ #2 / NM_002205.2 / Target:AACCTCACTTACGGCTATGTC
shRNA: GATCCGCCTCACTTACGGCTATGTCTTCAAGAGAGACATAGCCGTAAGTGAGG TTTTTTACGCGTG
ITGA5 _ #3 / NM_002205.2 / Target:AATGCCCTGAACCTCACTTTC
shRNA: GATCCGTGCCCTGAACCTCACTTTCTTCAAGAGAGAAAGTGAGGTTCAGGGCA TTTTTTACGCGTG
RhoA _ #1 / NM_001664.2 / Target: AAGGACCAGTTCCCAGAGGTG
shRNA: GATCCGGGACCAGTTCCCAGAGGTGTTCAAGAGACACCTCTGGGAACTGGTCC TTTTTTACGCGTG
RhoA _ #2 / NM_001664.2 / Target: AAGCAGGTAGAGTTGGCTTTG
shRNA: GATCCGGCAGGTAGAGTTGGCTTTGTTCAAGAGACAAAGCCAACTCTACCTGC TTTTTTACGCGTG
RhoA _ #3 / NM_001664.2 / Target:GGGTACATGGAGTGTTCAGCAAA
shRNA: GATCCGGTACATGGAGTGTTCAGCAAATTCAAGAGATTTGCTGAACACTCCAT GTACTTTTTTGCGGCCGCG
Supplementary Table 3: PRIMER SEQUENCES FOR RT-QPCR. Primers were designed with Primer3Plus software.
GENE / PRIMER SEQUENCE (with reference to CDS) (5’ to 3’) / AMPLICON SIZE (bp)hTBP / fw:TGCCCGAAACGCCGAATATAATC
rv:GTCTGGACTGTTCTTCACTCTTGG / 137
ITGA5 / fw:AAGAGCCG GATAGAGGACAAG
rv:AAGTGAGGTTCAGGGCATTC / 139
RhoA / fw:CAAGCATTTCTGTCCCAACG
rw:CTCTGCCTTCTTCAGGTTTCAC / 128