Total RNA from wild-type and Alpl knockout clones were extracted with the PureLink RNA Mini Kit (Thermo Fisher Scientific) 1 day after cells were plated, and complementary DNA was synthesized with SuperScript III reverse transcriptase (Thermo Fisher Scientific) from 1 μg of total RNA according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed with the StepOnePlus Real-Time PCR System (Applied Biosystems, MA, USA) and Fast SYBR Green Master Mix (Applied Biosystems). The expression level of each gene was normalized to both reference gene (Hprt1) and wild type. The primer sets for each target genes were as follows: Alpl, NM_007431 (forward) 5′-ctccaaaagctcaacaccaatg-3′ and (reverse) 5′-atttgtccatctccagccg-3′; Runx2, NM_009820 (forward) 5′-tggcttgggtttcaggttag-3′ and (reverse) 5′-ggtttcttagggtcttggagtg-3′; Sp7, NM_130458 (forward) 5′-ggagaccttgctcgtagatttc-3′ and (reverse) 5′-g cagagagacacccacagaaac-3′; Sparc, NW_009242 (forward) 5′-ggatgggttctgctctcatatt-3′ and (reverse) 5′-cctctgctctggccttaaatag-3′; Spp1, NM_009263 (forward) 5′-ctttcactccaatcgtccctac-3′ and (reverse) 5′-cagaaacctggaaactcctagac-3′; and Hprt1, NM_013556 (forward) 5′-ggccagactttgttggatttg-3′ and (reverse) 5′-cgctcatcttaggctttgtatttg-3′.

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