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Rat osteosarcoma cells as a therapeutic target model for osteoregeneration via
sclerostin knockdown
Bita Sedaghati1, Roomina Jahroomishirazi2, Annett Starke1, Michael C. Hacker1, Michaela
Schulz-Siegmund1
1
Pharmaceutical Technology, Institute of Pharmacy, University of Leipzig, Germany.
Department of Cell Techniques and Applied Stem Cell Biology, Center of Biotechnology and Biomedicine,
University of Leipzig, Germany
2
Fig.S1. SOST gene expression in growth medium (GM) vs. osteogenic medium (OM) after one
week in culture. Statistically significant differences between GM and OM are indicated by **
(p<0.01), n=4.
Fig.S2. Comparison of osteogenic stimulatory effects after silencing with four different siRNA
sequences (si-scl-1 to si-scl-4) in terms of ALP activity and mineralization in osteogenic medium
(OM). A: Absolute ALP activities on day 2 and 3 after seeding showed significantly increased ALP
in cultures treated with si-scl-1 and -3 over NC control, whereas ALP activity of cells treated with
si-scl-2 was significantly decreased. B: ALP activity normalized to cell number was significantly
increased in cells treated with si-scl-1, -2 and -3 compared to NC control. C: Mineralization
represented by calcium (µg/well) demonstrates no significant increase after SOST silencing. siscl-2 decreased the absolute calcium significantly compared to the non-coding control, on day 7.
D: At the same time point, mineralization normalized to cell number represented by calcium
(µg)/ 103cells, showed significantly increased mineralization for cells treated with si-scl-1, si-scl2 and si-scl-3 over NC control. E: Phase contrast images of cultures on day 7 confirmed lower
cell number and hence lower mineralization in cultures treated with si-scl-2 compared to other
groups. F: Cell number/ well on day 2 and 3 after seeding in osteogenic medium (OM).
Statistically significant differences compared to NC control are indicated by ** (p<0.01), n=4.
Fig. S3. Proliferation profile of UMR cells over 6 days in growth medium (GM), osteogenic
medium (OM), OM plus 100 ng/ml of BMP-2 (BOM-100) and OM with 250 ng/ml BMP-2 (BOM250). After 6 days, higher cell numbers were found in GM than in differentiating media.
Statistically significant differences between GM and differentiating media (OM, BOM-100 and
BOM-250) are indicated by ** (p<0.01), n=4.
Figure S4. ALP activity and cell number analysis in growth medium (GM) and osteogenic
medium (OM) on day 3. A: ALP activities were normalized to cell number. B: Cell numbers were
quantified by Picogreen DNA assay. Statistically significant differences compared to NC control
in relative media are indicated by ** (p <0.01), n=4.
Fig. S5. Effect of modified siRNA amounts (si-scl-1) on mineralization in cultures with high
seeding density (180,000 cells /cm2) treated with osteogenic medium (OM) on day 7. X: 3 µl
lipofectamin RNAimax and 3 pmol siRNA, Y: 6 µl lipofectamin RNAimax and 6 pmol siRNA, Z: 9 µl
lipofectamin RNAimax and 9 pmol siRNA. Statistically significant differences compared to NC
control are indicated as ** (p<0.01) and * (p<0.05), n=4.
A: Calcium amounts (µg) and cell number per well.
B: Calcium amounts (µg) normalized to 103 cells.