Figures & data
Figure 1 Cell viability 48 hours after transfection.
Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 1 Cell viability 48 hours after transfection.Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/1f005a53-927c-4148-8522-a03c536dc7f8/dijn_a_12190907_f0001_b.jpg)
Figure 2 (A and B) Images of MC3T3-E1 cells treated by different concentrations of LNPs/siRNA or lipo/siRNA 24 hours after transfection treatment. (C) Efficiency of cell uptake quantified by flow cytometry 48 hours after transfection.
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 2 (A and B) Images of MC3T3-E1 cells treated by different concentrations of LNPs/siRNA or lipo/siRNA 24 hours after transfection treatment. (C) Efficiency of cell uptake quantified by flow cytometry 48 hours after transfection.Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/d4a6caef-40af-4a7f-8911-7a4ba15c6939/dijn_a_12190907_f0002_b.jpg)
Figure 3 Expression of Noggin and Noggin protein in MC3T3-E1 cells transfected with different siRNA delivery systems. (A) Expression of Noggin on day-2 (PCR data). (B) Expression of Noggin protein on day-3 (data from Western blotting). (C) Semiquantitation of Noggin protein from Western blotting images analyzed by ImageJ software.
Notes: *P<0.05 and ***P<0.001 vs the blank group. ###P<0.001. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 3 Expression of Noggin and Noggin protein in MC3T3-E1 cells transfected with different siRNA delivery systems. (A) Expression of Noggin on day-2 (PCR data). (B) Expression of Noggin protein on day-3 (data from Western blotting). (C) Semiquantitation of Noggin protein from Western blotting images analyzed by ImageJ software.Notes: *P<0.05 and ***P<0.001 vs the blank group. ###P<0.001. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/d2638025-5ae6-49b9-b85b-d77a50c59029/dijn_a_12190907_f0003_b.jpg)
Figure 4 Effects of transfection via different systems on proliferation of MC3T3-E1 cells on days 1, 3, 5, and 7. The error bars represent the mean±SD (n=3).
Notes: *P<0.05 and **P<0.01 vs the blank group. #P<0.05, ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 4 Effects of transfection via different systems on proliferation of MC3T3-E1 cells on days 1, 3, 5, and 7. The error bars represent the mean±SD (n=3).Notes: *P<0.05 and **P<0.01 vs the blank group. #P<0.05, ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/a39c3d04-39e8-40bd-82f9-b59e4fb410db/dijn_a_12190907_f0004_b.jpg)
Figure 5 ALP activity (A) and relative expression of Runx2 mRNA determined by RT-PCR (B) in transfected MC3T3-E1 cells after 7 days and 14 days of culture in osteogenic medium.
Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000; ALP, alkaline phosphatase.
![Figure 5 ALP activity (A) and relative expression of Runx2 mRNA determined by RT-PCR (B) in transfected MC3T3-E1 cells after 7 days and 14 days of culture in osteogenic medium.Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000; ALP, alkaline phosphatase.](/cms/asset/93ad7340-c558-4527-8983-b14f16ae99cd/dijn_a_12190907_f0005_b.jpg)
Figure 6 Mineralization of transfected MC3T3-E1 cells on day 28. (A) Alizarin Red staining. Photos (upper) and microscopic images (lower) show formation of calcified nodules (stained red). (B) Quantification of calcified nodules dissolved by 10% cetylpyridinium chloride solution.
Notes: ***P<0.001 vs the blank group. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 6 Mineralization of transfected MC3T3-E1 cells on day 28. (A) Alizarin Red staining. Photos (upper) and microscopic images (lower) show formation of calcified nodules (stained red). (B) Quantification of calcified nodules dissolved by 10% cetylpyridinium chloride solution.Notes: ***P<0.001 vs the blank group. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/7a5124e8-ae77-470c-b0c7-56d1defd9455/dijn_a_12190907_f0006_c.jpg)
Figure 7 Effects of siNoggin and pBMP-2 treatment on expression of osteogenic-related proteins on day 3 (A, B) and 7 (C, D). (A, C) Representative Western blots of each protein. (B, D) Semiquantitative expression of BMP-2, OPN, β-catenin, and Runx2 proteins by ImageJ software.
Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. #P<0.05, ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 7 Effects of siNoggin and pBMP-2 treatment on expression of osteogenic-related proteins on day 3 (A, B) and 7 (C, D). (A, C) Representative Western blots of each protein. (B, D) Semiquantitative expression of BMP-2, OPN, β-catenin, and Runx2 proteins by ImageJ software.Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. #P<0.05, ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/4ef0cc11-2ab8-4f66-8291-02bfe3284f1a/dijn_a_12190907_f0007_b.jpg)
Figure 8 Effects of siNoggin and pBMP-2 treatment on expression of osteogenic-related proteins on days 14 (A, B) and 21 (C, D). (A, C) Representative Western blots of each protein. (B, D) Semiquantitative expression of β-catenin protein and Runx2 protein by ImageJ software. The error bars represent the mean±SD (n=3).
Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. #P<0.05, ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 8 Effects of siNoggin and pBMP-2 treatment on expression of osteogenic-related proteins on days 14 (A, B) and 21 (C, D). (A, C) Representative Western blots of each protein. (B, D) Semiquantitative expression of β-catenin protein and Runx2 protein by ImageJ software. The error bars represent the mean±SD (n=3).Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. #P<0.05, ##P<0.01 and ###P<0.001. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/4b60139a-ea92-4a80-8df3-8d33a14120b5/dijn_a_12190907_f0008_b.jpg)
Figure 9 LNPs/siNoggin promotes osteogenesis differentiation by activating BMP/Smad and GSK-3β/β-catenin signaling pathways in MC3T3-E1 cells on day-3. (A) Effects on the expression of phosphor-Smad/1/5/9 proteins. (C) Effects on the expression of phosphor-GSK-3β (Ser9) protein. (B, D) Semiquantitative expression of phosphor-Smad/1/5/9 or phosphor-GSK-3β (Ser9) proteins by ImageJ software.
Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. The error bars represent the mean±SD (n=3).
Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.
![Figure 9 LNPs/siNoggin promotes osteogenesis differentiation by activating BMP/Smad and GSK-3β/β-catenin signaling pathways in MC3T3-E1 cells on day-3. (A) Effects on the expression of phosphor-Smad/1/5/9 proteins. (C) Effects on the expression of phosphor-GSK-3β (Ser9) protein. (B, D) Semiquantitative expression of phosphor-Smad/1/5/9 or phosphor-GSK-3β (Ser9) proteins by ImageJ software.Notes: *P<0.05, **P<0.01 and ***P<0.001 vs the blank group. The error bars represent the mean±SD (n=3).Abbreviations: LNPs, lipopolysaccharide-amine nanopolymersomes; lipo, lipofectamine3000.](/cms/asset/835c0767-40c1-434f-8481-22bb068ae2bc/dijn_a_12190907_f0009_b.jpg)
Table S1 Primer sequences