3rd generation packaging mix

Human breast cancer cell lines T47D and MCF-7 were obtained from the ATCC and cell authentication was conducted at the ATCC using short tandem repeat DNA profiling. MISSION shRNA plasmids, shMEN1 (TRCN0000040141), shKMT2A (TRCN0000005956), shMED12 (TRCN0000018578), shWAPL (TRCN0000167548), and shGATA3 (TRCN0000019301), were obtained from Sigma-Aldrich (sequences listed in Table S2). Vehicle plasmid pLKO.1-puro (Addgene plasmid #8453) was obtained from Addgene. Plasmids were purified by QIAprep Spin Miniprep kit (Qiagen), and 293T cells were cotransfected with 3^rd Generation Packaging Mix (Applied Biological Materials, Richmond, BC, Canada) combined with either the shRNA plasmid or the vehicle plasmid for lentivirus production. The lentivirus-containing medium was harvested after 48 h of transfection, and cells were infected with lentivirus for 24 h. The infected cells were selected using puromycin at 4 μg/mL for 1 week. MI-503 (Selleckchem, Huston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock and diluted to the working concentrations in the medium.

PGAM1, NUDT7, shPGAM1, and shNudt7-containing lentiviruses were produced using the 3rd Generation Packaging Mix from the Applied Biological Materials, Inc. (ABM, LV053). Briefly, plasmids were transfected into Lenti-X 293 T cells (Clontech, 632180) using Lentifectin (ABM, G074) in OPTI-MEM I medium (ThermoFisher Scientific, 31985062) and cultured overnight. The supernatant was collected and lentiviral particles were concentrated using a Lenti-X Concentrator (Clontech, 631232) and stored at −80 °C. Cells were infected with lentivirus supernatant for 2 days in a humidified incubator at 37 °C in the presence of 5% CO₂. For in vivo articular cartilage delivery, concentrated lentivirus supernatant (1 × 10⁹ pfu) was injected into the intra-articular joint cavity of 8-week-old male mice once a week for 10 weeks.

The mouse HMGB1 gene was stably transduced to the knockout cells or wild-type cells using a Lentivirus system. Preparation of viral vector was as follows: 1 μg of a mouse HMGB1 lentiviral cDNA ORF clone, pLV-mHMGB1-GFPSpark (Sino Biological) and 1 μg 3rd Generation Packaging Mix (Applied Biological Materials) was transfected into 5 × 10⁵ 293 T cells (Takara Bio) using 6 μl TransIT-Lenti transfection reagent (Mirus Bio, LLC) at 37 °C for 48 h according to the manufacturer’s instruction. Recombinant virus was concentrated by Lenti-X Concentrator (Takara Bio), checked for its titer using the Lenti-X GoStix Plus (Takara Bio) and stored at − 80 °C until use. Lentiviral vector (3800 GoStix values compatible to 9.8 × 10⁴ infection units) was transduced into 4 × 10⁴ cells (MOI = 2.5) of knockout clones or WT cells with 8 μg/ml polybrene at 37 °C for 48 h. Then, the culture medium was changed to fresh medium not containing polybrene and the cells were diluted and put into a well of 96-well plate (0.2 cells/well) for single cell cloning. Expression of HMGB1-GFP fusion protein was confirmed by western blot analysis. After confirmation of HMGB1 expression, the cells were expanded and used for further study within two weeks of in vitro culture to avoid loss of HMGB1 expression.