Fig 1.
Influence of full length Smoc2 overexpression on differentiation and mineralization of MC3T3-E1 cells.
Gene expression (A) and Western blot (B) analysis of MC3T3-E1 cells overexpressing Smoc2 show successful overexpression of Smoc2. At D21 Smoc2+ cells show smaller increase in ALP activity (C) and less Alizarin red staining (D-E) compared to 3.1. (F) Smoc2+ modifies mRNA expression of Opn, Osx and Col1a2 during osteogenic differentiation. Statistically significant differences vs. D1 3.1 (internal control) are indicated as *: P<0.05; vs. D21 3.1 as #: P<0.05). Figures are representative of two independent experiments with three different Smoc2+ overexpressing clones, each experiment performed in triplicates. mRNA levels were normalized to S29.
Fig 2.
Influence of full length Smoc2 knock down on differentiation and mineralization of MC3T3-E1 cells.
Gene expression (A) and western blot (B) analysis of MC3T3-E1 cells with Smoc2 knock down show successful knockdown of Smoc2. At D21 ShSmoc2 cells show no difference in ALP activity (C) and Alizarin red staining (D-E) compared to control cells (Gipz). Statistically significant differences vs. D1 Gipz (internal control) are indicated as *: P<0.05; vs. ShSmoc2 at D1 as ¶: P<0.05; vs. D21 Gipz as #: P<0.05). Figures are representative of 3 independent experiments with 3 different ShSmoc2 clones, each experiment performed in triplicate.
Fig 3.
Influence of Smoc2 ΔCaBD on differentiation and mineralization of MC3T3-E1 cells.
Gene expression analysis of MC3T3-E1 cells overexpressing Smoc2 lacking the calcium binding domain shows successful overexpression (A). At D21 increase in ALP activity (B) is higher and Alizarin red staining (C-D) is stronger in ΔCaBD cells compared to Smoc2+. (E) mRNA expression of Osx, Opn, Col1a2 shows that ΔCaBD can decrease or suppress the influence of Smoc2 overexpression on MC3T3-E1 differentiation. mRNA levels were normalized to S29. Statistically significant differences vs. D1 Smoc2+ (internal control) are indicated as *: P<0.05; vs. ΔCaBD at D1 as ¶: P<0.05; vs. D21 Smoc2+ as #: P<0.05). Figures are representative for one experiment with 3 different Smoc2+ overexpressing clones performed in triplicate.
Fig 4.
Influence of calcium supplementation to Smoc2+ MC3T3-E1 cells on differentiation and mineralization.
Addition of extracellular calcium to Smoc2+ cells shows increased Alizarin Red staining (A-B) and a higher increase in ALP activity (C) at D21. Statistically significant differences vs. D1 Smoc2+ (internal control) are indicated as *: P<0.05; vs. Smoc2+ + 1mM Ca at D1 as ¶: P<0.05; vs. D21 Smoc2+ as #: P<0.05). Figures are representative for one experiment with 2 different Smoc2+ overexpressing clones performed in triplicate.
Fig 5.
Influence of Smoc2+ and of Smoc2 ΔCaBD on differentiation and mineralization of hPDCs and HUVECs.
(A) Confirmation of equal protein levels of secreted full length SMOC2 and SMOC2 ΔCaBD. (B-C) hPDCs treated with Smoc2+ supernatant show less Alizarin staining compared to 3.1, while ΔCaBD produces an intermediate response. Similar results were obtained for ALP activity (D). (E) HUVECs treated with Smoc2+ supernatant show less Alizarin staining compared to 3.1, ΔCaBD being intermediate as demonstrated by quantification of absorbance at 405 nm (F). Statistically significant differences vs. D1 (within same condition) are indicated as *: P<0.05; vs. 3.1 at D21 as ¶: P<0.05; vs. D21 Smoc2+ as #: P<0.05). Figures are representative of two independent experiments, performed in triplicate in hPDCs and of one experiment, performed in triplicate in HUVECs.