Role of VEGF in intermittent hypoxia-exposed neuroblastoma cells-mediated osteoclastogenesis in RAW 264.7 cells.

<p>(<b>A</b>) Intermittent hypoxia-exposed (IH) neuroblastoma cells were transfected with NTC siRNA (siNTC), HIF-1α-siRNA (siHIF-1α), HIF-2α-siRNA (siHIF-2α) or HIF-1α siRNA+HIF-2α siRNA(1∶1 ratio) mix (siHIF-1α+siHIF-2α) and cultured under normoxia. After 36 h, the cells were washed and maintained in serum-free medium for 24 h.VEGF concentration in cell culture supernatants was determined by ELISA. Values are expressed as mean ± SD (n = 3). °°P<0.01 parental (N) versus IH; * P<0.05, **P<0.01 IH/siNTC versus IH/siHIF-1α or IH/siHIF-2α-siRNA or IH/siHIF-1α+siHIF-2α. (<b>B</b>) RAW 264.7 cells were exposed to growth medium (control), growth medium with RANKL, CM from parental SH-SY-5Y cells (N), IH cells or IH cells transfected with NTC siRNA (siNTC), HIF-1α-siRNA (siHIF-1α), HIF-2α-siRNA (siHIF-2α) or HIF-1α+HIF-2α siRNA (1∶1 ratio) mix (siHIF-1α+siHIF-2α) and were allowed to differentiate under normoxia. The medium was changed after three days. After six days, cells were washed twice with PBS, fixed with 3.7% formaldehyde and stained for TRAP. RANKL-treated RAW cells served as a positive control. TRAP-positive multinucleated cells containing three or more nuclei were counted under a light microscope. Values represent the number of TRAP positive cells counted in ten different fields for each experiment from three independent experiments and expressed as mean ± SD. °°P<0.01 control versus RANKL; **P<0.01 IH/siNTC versus IH/siHIF-1α or IH/siHIF-2α or IH/siHIF-1α+siHIF-2α. (<b>C</b>) IH cells were transfected with NTC siRNA (siNTC) or HIF-1α-siRNA (siHIF-1α) and CM were collected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105555#s2" target="_blank">Methods</a>. Additionally parental, IH-siNTC, and IH-siHIF-1α cells were treated with VEGF antibodies and CM were collected. RAW 264.7 cells were exposed to growth medium (control), growth medium with RANKL, CM from parental or IH cells treated with NTC siRNA, HIF-1α-siRNA and/or VEGF antibodies and grown for six days with medium change after three days under normoxia. TRAP activity was assessed in cell lysates by the para-nitrophenyl phosphate hydrolysis assay. Results were normalized with total protein content of cultures and expressed as ηmol/min/mg protein. CM from RANKL-treated cultures served as a positive control. Values are expressed as mean ± SD (n = 4). °°P<0.01 control versus RANKL; **P<0.01 IH versus IH/VEGF Ab or IH/siHIF-1α.