vimentin overexpressed plasmid was purchased from Addgene 31922: p
vimentin-PSmOrange with full length of
vimentin sequence insertion [56 (
link)]. Plasmids of
vimentin knockdown were purchased from National RNAi Core Facility Platform in Taiwan:
shLuc: TRCN0000072244, ATCACAGAATCGTCGTATGCA,
shVim #3: TRCN0000029119, GCTAACTACCAAGACACTATT,
shVim #4: TRCN0000029121, GCAGGATGAGATTCAGAATAT.
Cells were transfected by different shVim sequences in a cell population and then selected by Puromycin for two weeks to generate stable clones for further experiment.
vimentin overexpressing stable clones were transfected in a cell population and then selected by Neomycin, followed by sorting to purify the
vimentin expressing cells.
siRNA were purchased from Thermo (L-003551-00-0005,
ON-TARGETplus SMARTpool, Human Vim) combined four siRNA sequences together:
J-003551-06, UCACGAUGACCUUGAAUAA;
J-003551-07, GAGGGAAACUAAUCUGGAU;
J-003551-08, UUAAGACGGUUGAAACUAG;
J-003551-09, GGAAAUGGCUCGUCACCUU.
And the control scramble siRNA sequence was (D-001810-01-05,
ON-TARGET plus Non-targeting siRNA #1): UGGUUUACAUGUCGACUAA
The primary antibodies used in the study, were as followed:
β1-integrin (BD),
GAPDH (Santa Cruz),
vimentin (Millipore),
vinculin (SIGMA),
paxillin (BD),
E-cadherin (BD),
β-catenin (BD), slug (Cell Signaling),
filamin A (Cell Signaling), and p-
filamin A (Cell Signaling).