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. 2009 Oct 9;150(11):4977–4988. doi: 10.1210/en.2009-0248

Figure 6.

Figure 6

Effect of Nox1 and Nox4 siRNAs on RANKL expression in murine primary osteoblasts. A, Analysis of siRNA knockdown efficiency. Osteoblasts were not transfected (UT) or transfected in complete medium with 100 nm siRNAs including Nox1, Nox4, or scrambled control (Dharmacon) for 12 h. Then cells were serum starved for 24 h before harvesting total RNA. RT-PCR was performed using specific murine Nox PCR primers or 18S as control. B, Effect of siRNAs on RANKL. Transfected cells were placed in SF medium for a total period of 48 h including a 24-h incubation time with or without CSF-1 (100 ng/ml) before determining the effect of each siRNA on RANKL mRNA expression. Bar graph shows semiquantitative analysis of siRNA treatment on RANKL mRNA expression by RT-PCR. c, P < 0.05 vs. scrambled control; d, P > 0.05 vs. siNox1+siNox4 alone. C, Analysis of siRNA transfection efficiency. Cells were not transfected (left panel) or incubated with Alexa Fluor 546-labeled negative control siRNA (right panel) as described in Materials and Methods, and nuclei were stained with DAPI. Composite overlay of DAPI and red fluorescence visualized by confocal microscopy is shown. D and E, Effect of siRNA knockdown on Nox proteins. Osteoblasts were placed in SF medium and not transfected (UT) or incubated with siNox1 (100 nm), siNox4 (200 nm) or scrambled control. After 24 h, cells were harvested and analyzed by RT-PCR (upper panels) and Western blot (lower panels). F, Effect of siNox1 on RANKL. Transfected cells in SF medium were incubated with or without CSF-1 (100 ng/ml) as described in B. Bar graph shows semiquantitative analysis of siRNA treatment on RANKL mRNA expression by RT-PCR. G, Quantitative real-time PCR for RANKL. Samples from the same experiment shown in F were analyzed by real-time PCR. Relative RANKL mRNA expression levels, normalized against 18S rRNA, were calculated using the 2−ΔΔCt method, and values were graphed as the mean expression level ± se. b, P < 0.01 vs. scrambled control without CSF-1. H, CSF-1 directly inhibits RANKL expression by osteoblasts. Scr., Scrambled control.