Effects of <i>Car9</i> siRNA on chondrocyte proliferation and expressions of marker genes of chondrocyte differentiation.

<p><i>Car9</i> siRNA or control siRNA was introduced into primary chondrocytes isolated from the rib cages of 1-day-old postnatal ddY mice. <b>A.</b> The expression of <i>Car9</i> mRNA was analyzed by real-time RT-PCR at 48 hours after introduction of <i>Car9</i> siRNA or control siRNA, with the level normalized to that of <i>Gapdh</i>. Data are expressed as the mean ± SD (n = 4) for fold changes caused by introduction of <i>Car9</i> siRNA. <i>P</i>-value obtained by two-tailed Mann-Whitney <i>U</i>-test (n = 4) indicated that the <i>Car9</i> siRNA used in this study significantly lowered the expression level of <i>Car9</i> mRNA (α = 0.05). <b>B.</b> Expression of CA IX protein in chondrocytes was detected by immunocytochemical staining at 48 hours after introduction of control siRNA (left 2 panels) or <i>Car9</i> siRNA (right 2 panels). Lower 2 panels show the results obtained without primary antibody against CA IX. Bar, 50 µm. <b>C. </b><i>Car9</i> siRNA (unfilled square) or control siRNA (filled square) was introduced to chondrocytes on day 0. Proliferation of chondrocytes was assessed spectrophotometrically using CellTiter 96^® Aqueous One Solution. Data are expressed as the mean ± SD (n = 4). At each time point, Mann-Whitney <i>U</i>-test with Bonferroni correction was performed to evaluate the difference between control siRNA- and <i>Car9</i> siRNA-introduced cells. No significant difference between the values was indicated at any time point (α = 0.01). <b>D.</b> At 48 hours after introduction of siRNAs, expressions of mRNAs for <i>Car9</i>, <i>Col2a1</i>, <i>Col10a1</i>, <i>Vegfa</i>, and <i>Mmp13</i> were analyzed by RT-PCR. <b>E-G.</b> The expressions of <i>Col2a1</i> (<b>E</b>), <i>Acan</i> (<b>F</b>), and <i>Col10a1</i> (<b>G</b>) were quantitatively analyzed by real-time RT-PCR. The expression level of each gene was normalized to that of <i>Gapdh</i>. Data are expressed by boxplots (n = 4) in the fold change by introduction of <i>Car9</i> siRNA (the sample maximum, the upper quartile, the median, the lower quartile, and the minimum observation). <i>P</i>-values determined by two-tailed Mann-Whitney U-test are indicated. <b>H.</b> At 4 days after introduction of <i>Car9</i> or control siRNA, chondrocyte cultures were stained with Alcian blue. Alcian blue bound to the cell matrix was extracted and determined spectrophotometrically (n = 4). A <i>P</i>-value determined by two-tailed Mann-Whitney <i>U</i>-test is indicated. Typical photographs are shown above the columns.