Figure 4. Constitutively elevated cAMP differentially affects primary osteoblasts and osteosarcoma cells.

(A) Primary osteoblasts treated with DMSO or forskolin for 72 hr and assessed for expression of Sca-1, CD51, PDGFRα , representative results shown, n=3 independent experiments. (B) Quantitation of cell surface markers from each treatment (n=3 independent cultures) (C) Expression of Sp7 (Osterix) and Runx2 by qPCR after 72 hr of forskolin treatment. Expression levels normalized to β2m; mean ± SEM (n=3). (D) Expression level of indicated genes over 21 days of treatment with DMSO or forskolin. Expression normalized to β2m; mean ± SEM (n=3). (E) Mineralisation analysis of primary osteoblasts at day 21 after treatment. Images are representative of 3 independent experiments. (F) Fibroblastic OS cells were treated with DMSO or forskolin for 72 hr and assessed for expression of Sca-1, CD51, PDGFRα, representative results shown. (G) Quantitation of cell surface markers from (n=3 independent cultures of fibroblastic OS) from each treatment. (H) Expression of Sp7 (Osterix) and Runx2 in fibroblastic OS by qPCR following 72 hr treatment. Expression levels normalized to β2m; mean ± SEM (n=3). (I) Expression of indicated genes in fibroblastic OS over 21 days from each treatment. Expression normalized to β2m; mean ± SEM (n=3). (J) Representative images of alizarin red stained fibroblastic OS cells treated with DMSO or forskolin for 21 day; n=3 independent OS cultures; *p<0.05, **p<0.001, ***p<0.0001. See Figure 4—figure supplement 1 and Figure 4—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.13446.012

Figure 4—figure supplement 1. cAMP has different effects in OS and primary osteoblasts.

(A, B) Expression of Runx2 and Osterix by qPCR and normalized to β2m; means ± SEM (n=3). (C, D) Quantitation of elution of Alizarin red stain from primary osteoblasts and fibroblastic OS cells treated with DMSO or 10 μM forskolin for 21 days. The data is representative of 3 independent experiments with each. (E) Cell surface profiling for Sca-1 and CD51 (αV Integrin), PDGFRα on osteoblastic OS cells (F) Quantitation of Sca-1/CD51, Sca-1/PDGFRα and CD51/PDGFRα populations in osteoblastic OS (n>3) with (blue bars) and without (black bars) forskolin treatment. (G) Mineralisation assay showing alizarin red staining of osteoblastic OS cells. (H) Quantitation of elution of Alizarin red staining of osteoblastic OS cells treated with DMSO or 10 μM forskolin for 21 days. The data is representation of 3 independent experiments. (I) Representative western blot for CREB1 following infection with 2 independent shRNA or control shRNA (shLuc). (J) Gene expression analysis of fibroblastic OS infected with the indicated shRNA and placed in differentiating conditions for the indicated periods of time. qPCR data normalized to β2m (n=3/cell type). (K-L) Representative alizarin stained wells and quantitation of alizarin elution from day 21 cultures in differentiating conditions with fibroblastic OS. For all the experiments above Students t-test was used, *p<0.05, **p<0.001, ***p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.13446.013

Figure 4—figure supplement 2. Level of cAMP in primary osteoblasts and osteosarcoma cells +/- forskolin.

(A) Intracellular cAMP levels in primary osteoblasts and fibroblastic OS cells at day 0 and day 21 of culture in differentiation inductive conditions with no exogenous agonist added (IBMX treated only); data expressed as cAMP level (pmol) per μg protein; graphed as mean ± SEM (n=3). (B) Intracellular cAMP levels in primary osteoblasts and fibroblastic OS cells at day 21 of culture in differentiation inductive conditions with forskolin (continuously present in the culture); data expressed as cAMP level (pmol) per μg protein; graphed as mean ± SEM (n=3). (C) Intracellular cAMP levels in primary osteoblasts and fibroblastic OS cells at day 21 of culture in differentiation inductive conditions with acute forskolin treatment at day 21 (20 min exposure to forskolin only); data expressed as cAMP level (pmol) per μg protein; graphed as mean ± SEM (n=3).

DOI: http://dx.doi.org/10.7554/eLife.13446.014