TABLE 3.
Effects of different treatments on pericellular HA coats
Coats were visualized using a particle exclusion assay in Hyal2 overexpressing (BB16Hy2+) cells and in control (BB16 and BB16mock) cells and expressed as the ratio of combined cell and pericellular matrix areas relative to the cell area. A value of 1.0 means no discernible coat. Results are presented as means ± S.E. of 17 to 81 cells in each group. p values in the last column result from comparisons of BB16mock and BB16Hy2+ cells in two-way analysis of variance with Bonferroni correction. Statistical analyses were performed in four separate settings: basal settings, coat inhibitors, potential Hyal2 activity inhibitors, and coat recovery after HA treatment. Statistical significance of the effect of treating either BB16mock or BB16Hy2+ cells with various agents was tested in one-way analysis of variance followed by Dunnett's test. The results are given in footnotes a–d. All other relevant comparisons in BB16mock and BB16Hy2+ cells were NS (not significant).
| BB16 | BB16mock | BB16Hy2+ | p | |
|---|---|---|---|---|
| Basal settings | ||||
| Untreated cells | 2.05 ± 0.05 | 2.08 ± 0.04 | 1.22 ± 0.07 | <0.001 |
| Scrambled RNA | 2.28 ± 0.11 | 1.09 ± 0.08 | <0.001 | |
| Hyal2 siRNA | 1.99 ± 0.07 | 1.71 ± 0.07a | NS | |
| Coat inhibitors | ||||
| Serum-free untreated cells | 2.01 ± 0.07 | 1.99 ± 0.05 | 1.18 ± 0.09 | <0.001 |
| HA10 oligomers, 100 μg/ml | 1.29 ± 0.02b | 1.11 ± 0.04 | NS | |
| HA6 oligomers, 100 μg/ml | 1.17 ± 0.09b | 1.07 ± 0.08 | NS | |
| Streptomyces HA (5 units/ml) | 1.08 ± 0.14 | 1.11 ± 0.05b | 1.14 ± 0.10 | NS |
| 4-MU, 100 μm | 1.21 ± 0.09 | 1.26 ± 0.06b | 1.19 ± 0.07 | NS |
| Potential Hyal2 activity inhibitors | ||||
| DMSO, 0.1% | 2.17 ± 0.07 | 1.08 ± 0.02 | <0.001 | |
| EIPA, 20 μm | 2.25 ± 0.08 | 1.86 ± 0.06c | NS | |
| CD44, transient overexpression | 2.14 ± 0.06 | 2.08 ± 0.05c | NS | |
| Coat recovery after HA treatment | ||||
| Coat recovery in 4 h | 2.08 ± 0.07 | 1.17 ± 0.04 | <0.001 | |
| Coat recovery with OX50 antibody | 1.37 ± 0.09d | 1.10 ± 0.03 | NS | |
| Coat recovery with OX50 and OX49 antibodies | 1.14 ± 0.08d | 1.06 ± 0.04 | NS | |
a p < 0.001 for Hyal2 siRNA versus scrambled siRNA in BB16Hy2+ cells.
b p < 0.001 for HA oligomers, HA, and 4-MU versus serum-free untreated BB16mock cells.
c p < 0.001 for CD44 transfection versus mock transfection and for EIPA versus DMSO vehicle in BB16Hy2+ cells.
d p < 0.001 for OX50 and OX50 plus OX49 versus controls in BB16mock cells previously treated with HA (coat recovery).