Galea GL et al, J Biol Chem Supplementary Figure 1 Supplementary Figure 1: Characterisation of long bone osteoblastic cells derived from mouse long bone diaphyses. A) clbobs treated with or without differentiation medium were fixed at the indicated time points and their alkaline phosphatase (ALP) activity determined relative to protein content as described in the supplementary methods. Bars represent the mean ±SEM, n = 6. ** p < 0.01, *** p < 0.001 versus the respective control group. B) ALP Fast Red staining of clbobs not treated with differentiation medium. C) Alizarin red staining of clbob cultures treated with or without differentiation medium for 21 days indicating the formation of mineralized nodules.
Supplementary Figure 2 Supplementary Figure 2: Confluence increases Saos-2 Sost expression. Sost expression was quantified by qrt-pcr in sub-confluent (~80% confluent) versus fully confluent cultures of Saos-2 cells. **p<0.01.
Supplementary Figure 3 Supplementary Figure 3: Saos-2 cells express and secrete sclerostin protein. Confluent Saos-2 cells were cultured as described in the methods, but for this experiment were not serum depleted. A) Sclerostin protein demonstrated by western blotting of cell lysates. Recombinant human sclerostin is shown as a positive control and actin as a loading control. B) Sclerostin levels in the cells supernatant was determined by ELISA as previously described (1), n = 6. Human serum sclerostin levels previously reported by Modder et al (2) in men and women are indicated.
Supplementary Fig 4 Supplementary Figure 4: The ERβ agonist ERB also down-regulates Sost expression. Saos-2 cells were treated with 0.1 μm ERB and harvested 8 hrs later. Sost expression was quantified by qrt-pcr. Bars represent means ± SEM. ***p<0.001.
Supplementary Figure 5 Supplementary Figure 5: Inhibition of ERα and ERβ prevents Sost down-regulation by E2. Saos-2 cells were treated with 0.1 μm following 16 hr pre-treatment with 1 μm ICI and harvested 8 hrs later. Bars represent the mean ± SEM. ** p < 0.01 versus vehicle control and ## p < 0.01 versus the E2-treated vehicle group.
Supplementary Figure 6 Figure 6: 10 μm PD98059 effectively inhibits ERK phosphorylation without altering Saos-2 viability. Saos-2 cells were treated with 10 μm PD98059 and fixed 24 hrs later to A) determine cell number and B) assess PI positivity as a measure of viability. Neither cell number nor the proportion of cells able to absorb PI (non-viable cells) was significantly altered by PD98059 treatment. In order to confirm that 10 μm PD98059 was able to inhibit ERK phosphorylation in our model, cells were treated with vehicle (veh.) or PD98059 and harvested 30 mins later. C,D) PD98059 significantly reduced ERK1/2 phosphorylation (p- ERK1/2) relative to total ERK as is illustrated by a representative western blot and quantified, n = 3. Tubulin is shown as a loading control. ** p < 0.01.
Supplementary methods: Characterisation of cortical long bone-derived mouse osteoblasts For characterisation studies, passage 1 clbobs were seeded at 25,000 cells/cm 2 and treated with or without 50 μm ascorbic acid and 10 mm β-glycerol phosphate in complete medium (differentiation medium). Cells were fixed in ice-cold methanol for 5 mins and alkaline phosphatase activity determined using the p-nitrophenyl phosphate Sigma Fast TM (Sigma, Dorset, UK) substrate conversion kit according to the manufacturer s instructions. Substrate conversion was normalized relative to total protein content using the crystal violet method following protocols previously-reported by other groups (3-4). Alkaline phosphatase activity was visualized using the naphthol AS-BI method with Fast Red (Sigma, Dorset, UK), as previously described by our group applied to similarly-derived rat cells (5), in cultures of mouse clbobs fixed in 4% paraformaldehyde solution, ph 7.4, for 20 mins at room temperature. Mineralized nodules in cultures treated with or without differentiation medium for 21 days were fixed in ice-cold methanol on ice for 5 mins, air dried, washed in phosphatebuffered saline, stained in 2% alizarin red solution (Sigma, Dorset, UK) ph 4.2 for 5 mins, and cleared under running water. Determining the effect of confluence on Saos-2 Sost expression Cells were seeded at 20,000 cells/cm2 or 40,000 cells/cm2 in 6-well dishes and cultured for 3 days so as to achieve different levels of confluence (sub-confluent or fully-confluent). Cells were harvested and RNA extracted for quantification of Sost expression by qrt-pcr. Determining the effect of the ERβ agonist ERB 041 on Sost expression ERB 041 (ERB) (6) was obtained from Tocris Bioscience (Bristol, UK) and reconstituted in ethanol. Saos-2 cells were cultured following the Sost regulation protocol, treated with 0.1 μm ERB and harvested 8 hrs later. Sost expression was quantified by qrt-pcr. Determining the effect of 24 hrs PD98059 treatment on cell viability and ERK phosphorylation Saos-2 cells were seeded at an initial density of 10,000 cells/well in 24-well plates and allowed to settle for 24 hrs before being serum-depleted in 2% charcoal-dextran stripped serum overnight. The next morning cells were treated with 10 μm PD98059 or vehicle and fixed 24 hrs later in buffered 4% paraformaldehyde, ph 7.6, which was then washed off in PBS. Cells were stained with DAPI such that cell number could be determined. Cell viability was determined by staining cells for with a 1:100 dilution of propidium iodide (PI), which stains non-viable cells (7), for 5 mins at room temperature. The number of cells and the proportion of cells stained positive for PI was determined using NIH Image J. To determine the effect of 10 μm PD98059 on ERK phosphorylation, Saos-2 cells were cultured following the protocol used to study Sost regulation. Cells were not serum-deprived in order to maintain detectable basal ERK phosphorylation, but instead were treated with PD98059 for 30 mins (the pre-treatment time normally used before exposure to strain or
treatment with agonists). Cells were then washed in glacial PBS and lysed in denaturing lysis buffer as previously described (8). ERK phosphorylation was analyzed by Western blotting with primary antibodies to phosphorylated (perk) and total ERK (Cell Signaling Technologies) using conventional resolution and transfer methods (8). Secondary, fluorescently labelled antibodies were from Li-Cor and the fluorescent signals were acquired and analyzed using Odyssey Fc dual-mode imaging system (Li-Cor) as recommended by the manufacturer. Band intensity of p-erk1/2 relative to total-erk1/2 was determined using NIH Image J.
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