Plasmids and generation of stable cell lines.

SOX9 small interfering RNA (siRNA) (catalog number D-059108-01), ALDH1A1 siRNA (catalog number L-008722-00-0005), nontargeting siRNA (catalog number D-001810-01), and pLKO.1 lentiviral plasmids for SOX9 knockdown (catalog number RHS4533-EG6662) were purchased from Dharmacon (Lafayette, CO). An empty pLKO.1 vector was used as a control, and the virus was produced in HEK293T cells (ATCC) as described previously (51). For SOX9 overexpression, SOX9 cDNA from pCMV-AC-GFP-SOX9 (Origene, Rockville, MD) was amplified by PCR and subcloned into pLUTzeo (a gift from A. Ivanov, West Virginia University, WV). Viral particles were produced as described above, and the empty vector was used as a control. siRNA transfections (50 nM) were performed on 6-well dishes with the Lipofectamine RNAiMAX transfection reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Cells were lysed or used for subsequent experiments at 48 h posttransfection. The short hairpin RNA (shRNA) and siRNA sequences are listed in Table S3 in the supplemental material. pcDNA3-HA-ALDH1A1 was a gift from Steven Johnson (Addgene plasmid number 11610) with a point mutation fixed by site-directed mutagenesis. pcDNA3-mRFP was a gift from Doug Golenbock (Addgene plasmid number 13032).