Lv5 lentiviral vector

The plasmids were constructed according to standard methods. All structures were verified by appropriate restriction digestion and/or sequencing. Human full-length SFRP2 cDNA was generated using a standard gene synthesis method and sub-cloned into the LV5 lentiviral vector (GenePharma Company). Full-length WNT1 cDNA fused to a hemagglutinin (HA) tag was generated using a standard gene synthesis method and sub-cloned into the pLNCX retroviral vector. Short hairpin RNAs (shRNAs) with complementary sequences of target genes were sub-cloned into the pLKO.1 lentiviral vector (Addgene). For viral infections, MSCs were plated overnight and then infected with retroviruses or lentiviruses in the presence of polybrene (6 μg/ml, Sigma-Aldrich) for 6 h. After 48 h, the infected cells were selected with 2 μg/ml puromycin. Scrambled shRNAs (Scramsh) were purchased from Addgene. The target sequence for the shRNAs is: SFRP2 shRNA (SFRP2sh), 5′-ttgatgtaggttatctccttc-3′.

The full-length human HHIP-AS1 constructs were created following standard protocols and verified by gene sequencing. The cDNA of HHIP-AS1 was subcloned into the LV5 lentiviral vector (GenePharma Company, Suzhou, China) for overexpression. Subsequently, the LV3 lentiviral vector (GenePharma) was used to construct a short hairpin RNA (shRNA) of HHIP-AS1. The PDLSCs were seeded and cultured in 100 mm dishes overnight and infected with lentiviruses and 6 μg/mL polybrene (Sigma-Aldrich) for 12 h. Then, the transfected PDLSCs were selected by proper antibiotics after 72 h of infection. The target sequences of the shRNAs were as follows: control shRNA (Consh), 5′-TTCTCCGAACGTGTCACGTTTC-3′, and HHIP-AS1 shRNA (HHIP-AS1sh), 5′-GCACCAATGCATCTTGTATGA-3′.

The plasmids were constructed with standard methods; all structures were verified by appropriate restriction digestion and/or sequencing. Human full-length XR-111050 cDNA from BMSCs was produced with a standard PCR protocol. This sequence was subcloned into the LV5 lentiviral vector (Genepharma Company, Suzhou, China). Short-hairpin RNAs (shRNA) with the complementary sequences of the target genes were subcloned into the LV3 lentiviral vector (Genepharma Company, Suzhou, China). For viral infections, MSCs were plated overnight and then infected with lentiviruses in the presence of polybrene (6 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) for 6 h. After 48 h, infected cells were selected with 1 μg/mL puromycin for 7 days. The target sequences for the shRNA were: LV3 shRNA (Consh), 5’-TTCTCCGAACGTGTCACGTTTC-3’; XR-111050 shRNA (XR-111050sh), 5’-GGACGTGTCTTTCAGGGAAAG-3’.