TABLE 3.

Measurement of HA-mediated CD44v3^highALDH1^high cell invasion

* Twenty four transwell units containing 5-μm porosity polycarbonate filters coated with the reconstituted basement membrane substance Matrigel were used for monitoring in vitro tumor cell invasion. Specifically, CD44v3^highALDH1^high cells [1 × 10⁴ cells/well-untransfected (treated with normal IgG or anti-CD44 antibody) or transfected with DOT1L siRNA, RhoC siRNA, cIAP-2 siRNA, XIAP siRNA, or scrambled siRNA or negative miRNA control or anti-miR-10b inhibitor] were placed in the upper chamber of the transwell unit in the presence of 50 μg/ml HA. The growth medium containing high glucose DMEM supplemented by 10% fetal bovine serum were placed in the lower chamber of the transwell unit. After 18h incubation at 37 °C in a humidified 95% air, 5% CO₂ atmosphere, cells on the upper side of the filter were removed by wiping with a cotton swab. Cell invasion processes were determined by measuring the cells that migrated and invaded to the lower side of the polycarbonate filters by standard cell number counting methods. The CD44-specific cell invasion was determined by subtracting non-specific cell invasion [i.e. cells that migrated (invaded) to the lower chamber in the presence of anti-CD44 antibody treatment]. The CD44-specific cell invasion in normal IgG/scrambled siRNA-treated cells without HA (A and B and D, control) or negative miRNA-treated cells without HA (C, control) is designated as 100%. The values expressed in this Table are presented as the means ± S.D. All assays consisted of at least six replicates and were performed on at least five different experiments.

^a Data are statistically significant (P < 0.005; analysis of variance; n = 5) as compared with control samples (normal IgG-treated cells without HA addition).

^b,d Data are statistically significant (P < 0.005; analysis of variance; n = 5) as compared with control samples (scrambled siRNA-treated cells without HA addition).

^c Data are statistically significant (P < 0.005; analysis of variance; n = 5) as compared with control samples (miRNA negative control-treated cells without HA addition).