Ncam1

An outgrowth method was utilized to culture synovial MPCs [9 (link), 10 , 14 (link)]. Upon receipt of synovium, each tissue explant was minced and seeded in a 24-well culture plate and incubated at 37 °C and 5% carbon dioxide (CO₂) with 1 mL of MesenCult™ (Stemcell Technologies) culture media added. Within 11 days post-seeding, outgrown cells were adherent to the plastic and reached 30–40% confluency. At this point, cells were gently dissociated via mechanical stimulation and seeded into a T-25 cell culture flask. Media was changed every 3 days. After cells reached 70% confluency, the cells were washed, resuspended, and subjected to magnetic-activated cell sorting (MACS) purification entailing hematopoietic lineage depletion (FCGR3A, CD19, CD3E, NCAM1, CD14, GYPA, FCGR3B, ITGA2B)(BD Biosciences). Purified cells were then expanded in T-75 flasks. Media was changed every three days and cells were passaged when 70–80% confluency was reached. Cells passaged a maximum of 3 times were used for flow cytometry phenotyping, multipotent differentiation analysis and proteomics.

The chemicals quercetin (Q4951; Sigma-Aldrich, ON, Canada) and ICG-001 (S2662; Selleck chemical, TX, USA) were dissolved in DMSO and added to the cells 15h before co-treatment with TGFB1. Unless indicated otherwise, human recombinant TGFB1 (240-B; R&D Systems, MN, USA) was added to cells at a final concentration of 6.4 ng/ml for 48h.
The plasmids coding for ST8SIA2 (MR205823) and ST8SIA4 (MR205502) were purchased from Origene (MD, USA). Transient knockdowns were achieved with SilencerSelect siRNAs against Prnp (s72188; Life Technologies) and Ctnnb1 (s63418; Life Technologies). ON-TARGETplus SMARTpools were obtained from GE HealthCare (ON, Canada) to target St8sia2 (L-042781-01-0005) and St8sia4 (L-044724-01-0005) transcripts.
Immunoblotting made use of antibodies against NCAM1 (1:6666, 556324 or 556325; BD Biosciences, ON, Canada), PrP (1:2000, A03213; Bertin Pharma, France), E-cadherin (1:4000, 3195; Cell signaling, MA, USA), p-CREB (1:1000, 9198; Cell Signaling), SNAI1 (1:1000, 3879; Cell Signaling) and Lamin A (1:1000, 26300; Abcam). For the microscopy analyses, the Alexa Fluor 488 goat anti-rabbit IgG (1:200, A31627) and Alexa Fluor 647 phalloidin (1:200, A22287) antibodies were purchased from Life Technologies (ON, Canada).