RESUMEN
Objective To develop a new set of algorithms for high-resolution cellular traction force recovery based on two-dimensional Fourier domain by addressing the ill-posed nature of classic cellular force traction recovery. Methods By exploring the inherent characteristics and rules of displacement data on the substrates and Green’s function in the Fourier domain, the phenomenon of ill-posed deconvolution arising in cellular force traction recovery was investigated and a set of self-adaptive filtering algorithms was consequently developed to remarkably restrain the high frequency noise amplification. Results The ill-posed nature of classical Fourier transform traction cytometry (FTTC) made cellular traction force recovery extremely unstable, especially for relatively dense displacement data sampling. In contrast, the proposed self-adaptive filtering algorithms based on FTTC could make cellular traction force distribution more stable and reliable, as the effect of high frequency noise in displacement field on recovery results was weakened significantly. Conclusions This new technique for cellular traction force recovery can effectively suppress the noise and therefore improve the stability of force recovery procedure and spatial resolution, which is expected to find wider application in the study of cell substrate interactions.
RESUMEN
<p><b>BACKGROUND AND OBJECTIVE</b>iASPP, an inhibitory member of the apoptosis-stimulating proteins of p53 (ASPP) family, has been found to be up-regulated in various human tumor types. This study was to construct an efficient doxycycline-regulated, lentiviral vector-mediated knockdown system for iASPP that will allow for inducible down-regulation of iASPP gene expression and preliminary functional analysis.</p><p><b>METHODS</b>A pair of complementary oligos with hairpin structures targeting the iASPP gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The fragment containing the shRNA cassette was cloned to pLVCT-tTR-KRAB plasmid. The recombinant vectors were co-transfected with viral packaging mix into 293T cells, and viral supernatant was harvested to determine the titer. After treatment with or without doxycycline, HepG2 cells infected with virus were harvested and the expression of iASPP was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Its effects on tumor growth were characterized using MTS assay, soft agar colony formation, and flow cytometry analysis.</p><p><b>RESULTS</b>The lentiviral vector expressing shRNA that targets to the oncogene iASPP was constructed successfully. HepG2 infected with the lentivirus expressing shRNA against iASPP inhibited the expression of iASPP in the presence of doxycycline, which resulted in the repression of tumor cell proliferation and anchorage-independent growth potential.</p><p><b>CONCLUSIONS</b>The lentiviral vector-mediated tet-on system demonstrates efficient and inducible knockdown of iASPP in hepatocellular carcinoma cells. iASPP gene may be involved in tumorigenesis and progression of human tumors.</p>