Lentifectin

To visualize actin polymers in live cells after encapsulation in gel coatings while minimally impacting baseline cellular functions, the plasmid containing F‐tractin fused with the fluorescent gene tdTomato (a gift from Andrei Karginov, UIC) was cloned into the lentiviral vector pCW57.1 (a gift from David Root, Addgene plasmid #41393) for inducible expression in the presence of doxycycline. F‐tractin is known to selectively bind to polymerized F‐actin.^[58] Lentiviral particles were produced with a second‐generation lentiviral packaging system (LV003, Applied Biological Materials) using a transfection reagent (Lentifectin, Applied Biological Materials) in HEK293T cells. Lentiviral particles were purified and applied to D1 MSCs at passage 5 with polybrene (8 µg mL⁻¹; Sigma) for 3 days, followed by the selection of transduced cells by puromycin (5 µg mL⁻¹; Sigma) for 7 days and sorting of tdTomato⁺ cells after doxycycline (500 ng mL⁻¹; Cayman Chemical) treatment for 1 day.

Lentiviruses containing miR144-3p precursor (miR144-3p), miR144-3p specific siRNA (anti-miR-144-3p) and negative control lentivirus (Applied Biological Materials Inc., Richmond, BC, Canada) were packaged using the 3rd Generation Packaging Mix (Applied Biological Materials) and transduced into the human 293FT host cell line using Lentifectin™ (Applied Biological Materials) in Opti-MEM^® I medium (Invitrogen). The supernatant was collected, and lentiviral particles were concentrated using the Lenti-X Concentrator (Clontech, Mountain View, CA, USA).

LentimiRa-Off-miR-126-5p vector was purchased from Applied Biological Materials, Inc. (ABM, Vancouver, BC, Canada; mh30100). Plasmid DNA was transfected into Lenti-X 293T cells (Clontech, Palo Alto, CA, USA; #632180) using Lentifectin (ABM, #G074) and a third-generation packaging mix (ABM, #LV053) in serum-free DMEM, and FBS was supplemented after 6–8 h. The supernatant contained with lentiviral particles was concentrated using a Lenti-X Concentrator (Clontech, #631232) and stored at −80 °C. For in vivo delivery, concentrated lentivirus (1 × 10⁹ PFU) was injected into the intra-articular joint cavity every week for eight weeks.

For stable expression of STARD13-3′UTR, CDH5-3′UTR, HOXD1-3′UTR, and HOXD10-3′UTR, sequences of 3′UTRs were subcloned into pLVX-ZsGreen and referred as pLVX-ceRNAs-3′UTR. shRNA oligos were synthesized by Sangon Co., Ltd. After annealing, double-strand oligos were inserted to lentiviral pLKO.1-Puro vector (Addgene). To package lentivirus, HEK-293T cells were co-transfected with the lentiviral vector described above and packaging vectors psPAX2 and pMDG.G using Lentifectin (ABM, USA). Cells were infected with the virus in the presence of 2 μg/ml polybrene. The infected cells were selected with puromycin (Sigma, 2 μg/ml) for 2 weeks. After two rounds of infection, qRT-PCR and Western blot analyses were used for verification. Meanwhile, cells infected with pLVX-ceRNAs-3′UTR were selected by fluorescent cell sorting.
The 3′UTRs of LATS1 and LATS were cloned into the luciferase reporter vector (pMIR-Report, Ambion, Carlsbad, CA, USA), and the corresponding plasmids were denoted as pMIR-LATS1-3′UTR and pMIR-LATS2-3′UTR. Sequences of primers used for plasmid construction in this study were listed in Additional file 4: Table S4. All constructs were confirmed by DNA sequencing.