Figure 4.

Validation that IL-9 induced six differentially expressed genes during osteoclast differentiation. (A–F) Cells derived from peripheral blood (PB) of healthy control (HC) and cells derived from PB and synovial fluid (SF) of patients with RA were treated as indicated with macrophage colony-stimulating factor (M-CSF; 25 ng/mL), soluble receptor activator of nuclear factor κB ligand (sRANKL; 50 ng/mL), or IL-9 (100 ng/mL) for 4 days. Cells were then lysed followed by RNA extraction and cDNA preparation from an equal quantity of RNA. Glyceraldehyde phosphate dehydrogenease (GAPDH) was used as an internal control for validation of the differentially expressed genes, as the expression of GAPDH showed least variation with stimulation. Quantitative real-time PCR (RT-PCR) was performed for ephrinB2 (EFNB2), ATP-binding cassette subfamily A member 7 (ABCA7), Krueppel-like factor 2 (KLF2), cadherin 6 (CDH6), acyl-coenzyme A synthetase (ACSM4), and potassium voltage-gated channel subfamily A member 3 (KCNA3). The graphs represent the relative expression of these genes (mean  ±  SD; n  =  8). Statistical analysis was performed using paired Student’s t-test comparing M-CSF/sRANKL/rIL-9-treated cells to M-CSF/sRANKL-, M-CSF-, and M-CSF/sRANKL-treated cells to M-CSF (*: p  ≤  0.05; **: p  ≤  0.005; ***: p  ≤  0.0005; ns–non significant).