SUPPLEMENTARY METHODS
Gene reporter assay. For luciferase reporter assay, cells were transiently transfected with
Exgen500
(Euromedex,
Mundolsheim,
France)
according
to
the
manufacturer’s
recommendations. pAP1-Luc (Stratagene; Agilent Technologies, Basel, Switzerland) and
phRL-SV40 (Renilla expression vector; BioVisualTech, Bellevue, WA, USA) plasmids were
added at a concentration of 2 µg/well for 6-well plates. Transcriptional activity was evaluated
using a Luciferase Assay Kit (Promega, Charbonière, France) and Renilla reporter assay kit
(Roche, Meylan, France) according to the manufacturer’s instructions. All assays were
performed in 5-6 replicates and the data are presented as means ± S.D.
Quantitative PCR amplification. Primers sequences for the various markers of osteoblast
differentiation (Runx2, osterix, type I collagen and alkaline phosphatase) are: Runx2 sense 5’TCTGGCCTTCCACTCTCAGT-3’ forward 5’-GACTGGCGGGGTGTAAGTAA-3’; Osterix
sense 5’-CATCTGCCTGGCTCCTTG-3’ forward 5’-CAGGGGACTGGAGCCATA-3’; ALP
sense 5’-GGACATGCAGTACGAGCTGA-3’ forward 5’-CCACCAAATGTGAAGACGTG3’;
Col1A1
sense
5’-AGCCAGCAGATCGAGAACAT-3’
forward
5’-
CGCCATACTCGAACTGGAAT-3’.
Alkaline phosphatase activity and osteogenesis. ALP activity was determined using the
Alkaline Phosphatase kit (BioRad Laboratories). ALP cytochemical detection was performed
using Sigma FAST BCIP/NBT kit (Sigma-Aldrich) and microphotographed using an
Olympus microscope (Japan). All assays were performed in 6-8 replicates and the data are
presented as means ± S.D.
For in vitro matrix mineralization, the medium was supplemented with 50 µg/ml ascorbic acid
and 3 mM NaH2PO4. Calcium deposition onto extracellular matrix was evaluated by alizarin
red staining (2% in water, pH 4.2). After digital scanning, mineral content was extracted using
hexadecylpiridium chloride (10 mM in sodium phosphate buffer) and quantified by
1
spectrophotometry at 562 nm. Assays were performed in 4 replicates and the data are
presented as means ± S.D.
SUPPLEMENTARY FIGURES LEGENDS
Supplemental Data 1. MT2A overexpression modulates intracellular signaling pathways.
A/ Phosphorylation status of ERK1/2-MAPKs, p38, JNK and Akt were evaluated by western
blot in SaOS2 and U2OS cells overexpressing or not MT2A. Ratios were corrected for actin
content. B/ The AP1-dependent transcriptional activity was evaluated in SaOS2 and U2OS
cells using an AP1-cis reporting luciferase assay. Results are expressed as treated over control
ratio (mean ± SEM). *: P<0.05 vs. parental cells.
Supplemental Data 2. MT2A overexpression promotes osteoblastic differentiation of
osteosarcoma cells. a/ Runx2, Osterix, Type I Collagen and Alkaline Phosphatase mRNA
levels were evaluated by quantitative RT-PCR in SaOS2 or U2OS parental and MT2A
overexpressing cells. b/ ALP activity was measured at different time points in SaOS2 and
U2OS cells overexpressing or not MT2A. c/ ALP activity was evaluated by colorimetric
assay. d/ Matrix mineralization was evaluated by von Kossa staining. e/ Mineral content was
measured by colorimetric assay. Results are expressed as treated over control ratio (mean ±
SEM). *: P<0.05 vs. parental cells.
Supplemental Data 3. MT2A silencing reduces osteoblastic differentiation of
osteosarcoma cells. a/ Runx2, Osterix, Type I Collagen and Alkaline Phosphatase mRNA
levels were evaluated by quantitative RT-PCR in SaOS2 or U2OS parental and MT2A
silenced cells. b/ ALP activity was measured at different time points in SaOS2 and U2OS
cells silenced or not for MT2A. c/ ALP activity was evaluated by colorimetric assay. d/
Matrix mineralization was evaluated by von Kossa staining. e/ Mineral content was measured
2
by colorimetric assay. Results are expressed as treated over control ratio (mean ± SEM). *:
P<0.05 vs. control cells.
3