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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: J Bone Miner Res. 2018 Mar 23;33(6):1166–1182. doi: 10.1002/jbmr.3409

Figure 4: MLO-A5 cell lines stably expressing GFPtpz-collagen and mCherry-collagen.

Figure 4:

A) MLO-colCherry cell line stably transfected with mCherry-collagen construct D and MLO-colGFP cell line stably transfected with GFPtpz-collagen construct E. Note the bright mCherry or GFPtpz fluorescence that remains intracellular without ascorbate (without AA) and forms a fibrillar collagen matrix with ascorbate (with AA). Insets show untransfected parental MLO-A5 cells for comparison (bar = 50µm). B) Views of the upper and lower surface of a 3D rendered confocal image of MLO-colGFP cell cultures in which the nuclei are stained with DAPI (blue) and the cell membrane is stained with DiD (red). Note that the majority of the collagen (green) is deposited at the lower surface of the cell layer. The pair of images on the right show a vertical slice through the 3D rendered image, confirming that the collagen is deposited below the cell layer (i.e. at the basal surface of the cells). Bar = 50µm. C) Immunogold EM localization of mCherry-collagen in banded collagen fibrils in MLO-colCherry cells using anti-DsRed(mCherry) antibody detected with 6nm gold-anti-rabbit antibody. Note the specific labeling of banded collagen fibrils and lack of background signal in the IgG control (bar = 200nm). Insets show enlargement of boxed areas. D) Co-IP of α1(I) collagen with mCherry-tagged α2(I) collagen. Conditioned media or a salt extract of the ECM was immunoprecipitated with anti-DsRed(mCherry) antibody, then immunoblotted with LF67 antibody against collagen α1(I). Note the specific band at ~120–125kDa (red arrow) detected only in MLO-colCherry cells and not untransfected controls. The band is only pulled down with anti-DsRed (mCherry) and not with control IgG. Higher MW bands in which the N or C terminus have not been fully processed are seen in the conditioned media, indicated by black arrows. Numbers to the right indicate molecular weight markers (kDa). [Images are representative of ≥3 experiments in A), B) & C) and 4 experiments with media/ 2 experiments with salt extract in D)].