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. 2014 Aug 22;9(8):e105555. doi: 10.1371/journal.pone.0105555

Figure 1. Effects of intermittent hypoxia exposure to neuroblastoma cells on in vitro osteoclastogenesis.

Figure 1

RAW 264.7cells were supplemented with CM from the SH-SY5Y neuroblastoma cells and characterized for the osteoclastogenic response. (A) RAW 264.7cells were exposed to growth medium alone (control), growth medium with RANKL (100 ηg/ml) or CM from parental SH-SY5Y cells (N), intermittent hypoxia exposed (IH) cells, or stable transfectants (HIF-1α, shLuc and shHIF-1α) and were allowed to differentiate for six days under normoxic condition. The medium was changed after three days. CM from knockdown cells (shLuc and shHIF-1α) that were grown under hypoxia (1% O2, 24 h) were also evaluated. Cells were washed twice with PBS, fixed with 3.7% formaldehyde and stained for TRAP with Acid Phosphatase, Leukocyte (TRAP) kit according manufacturer's instructions. A representative image is shown. Arrow indicates cells multinucleated and TRAP-positive. (B) Multinucleated and TRAP-positive cells in each well were counted. Graphical representation of the number of osteoclast-like cells is shown. Values represent 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<0.05, **P<0.01 Parental (N) versus IH or HIF 1α; # P<0.05 shLuc/normoxia versus shLuc/hypoxia or shHIF 1α/hypoxia. (C) RAW 264.7cells were exposed to growth medium (control), growth medium with RANKL (100 ηg/ml) or CM from parental SH-SY5Y cells (N), IH cells, stable transfectants (HIF-1α, shLuc and shHIF-1α), or knockdown cells (shLuc and shHIF-1α) that were exposed to hypoxia (1% O2, 24 h) and were grown under normoxic condition for six days. The medium was changed after three days. Cells were assessed by real RT-PCR for the expression of TRAP, CaSR and Cath K mRNAs. Values are from three independent experiments in triplicates and expressed as mean ± SD. °°P<0.01 control versus RANKL; * P<0.05, **P<0.01 Parental (N) versus IH or HIF 1α; # P<0.05, ##P<0.01 shLuc/normoxia versus shLuc/hypoxia or shHIF 1α/hypoxia.