Skip to main content
. 2022 Jan 19;17:457–470. doi: 10.1016/j.bioactmat.2022.01.019

Fig. 2.

Fig. 2

Osteogenic differentiation capacity of conditioned media from Nell1/BMSCs (Nell1/CM)depends on EVs. (A) Representative images of Nell1-green fluorescent protein (GFP) adenovirus (Ad-Nell1) infection in BMSCs in vitro. (B) Immunofluorescence-based detection of NELL1. (C) Determination of Nell1 overexpression in BMSCs by RT-qPCR. (D) RT-qPCR analysis of Nell1 transcription in BMSCs infected with Ad-Nell1 at day 2, 6, and 9. (E) Alkaline phosphatase (ALP) staining and quantitative ALP assay of BMSCs incubated with Nell1/CM or Ctrl/CM for 7 days. (F) Alizarin red staining and semi-quantitative analysis of BMSCs incubated with Nell1/CM or Ctrl/CM for 14 days. (G) RT-qPCR determination of osteogenic markers expression on day 7 and 14. (H) Quantification of EV protein content. EVs were collected from the conditioned media of each group: Ctrl/BMSCs, Nell1/BMSCs treated with or without GW4869. (I) Mineralization induction in fresh osteoinductive medium with Nell1/CM, Ctrl/CM, or Nell1-GW4869/CM. RT-qPCR determination of osteogenic marker expression on day 2, 6, and 9. (J) In co-incubation mineralization experiments, ALP staining and quantitative ALP assay at day 14. (K) Alizarin red staining of BMSCs and semi-quantification at day 21. *p < 0.05, **p < 0.01, ***p < 0.001, NS, non-significant, via t-test or one-way analysis of variance, n = 3. CM, conditioned media.