Ultra low attachment microplate

For spheroid formation assays, single cell suspensions from HCC1 and HCC2 were generated using FACS and 1.5 × 10⁴ HCC1 cells or 1.0 × 10⁴ HCC2 cells were seeded in 6-well Ultra-Low Attachment Microplates (Corning, Corning, NY). The number of spheroids was determined 21 days after seeding.

The unanchored growth ability was detected by sphere formation assay. Briefly, 2000 cells transfected with DARS-AS1 shRNAs or control shRNAs were cultured on ultra-low attachment microplates (Corning), with the media changed every 4 days. Spheres were counted after 14 days. For the gain-of-function assay, 500 cells transfected DARS-AS1-overexpressing plasmid or control plasmid were used instead and otherwise the methodology was unchanged.

3000 single cells were seeded into 24-well Ultra-Low Attachment Microplates (Corning, USA) in serum-free DMEM/F12 (Invitrogen, USA), supplemented with B27 (1:50, Invitrogen, USA), 20 ng/ml EGF (Peprotech, USA), 10 ng/ml bFGF (Invitrogen), 4 μg/ml insulin (Sigma, USA) and 20% methylcellulose (Sigma, USA). Spheres were collected 7 days afterwards.

Single-cell suspensions of 1000 cells were seeded in 6-well Ultra-Low Attachment Microplates (Corning, Corning, NY) for spheroid assays. The number of spheroids was measured 12 days after seeding.
For flow cytometry analysis, cultured cells were trypsinized, washed, and resuspended in phosphate-buffered saline plus 0.5% bovine serum albumin. They were incubated with Allophycocyanin (APC)-conjugated antibodies on ice for 20 mins in the dark. Data were collected with a FACS Calibur flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). The detailed information for all antibodies is listed in Supplementary Table S2.

A 2 ml sample of CM spheroids were treated with CM dissociation media (CMDM) (STEMCELL Technologies) for 15 min at 37°C with periodic mixing then counted to determine culture density. ECs, SMCs, and CFs were dissociated from monolayer cultures and 5.0 × 10⁶, 2.5 × 10⁶, and 2.5 × 10⁶ of each respectively were combined and resuspended in 1 ml of spheroid media (OM) (Supplemental Table S3). Based on cell counts 1.0 × 10⁷ total CMs were collected as whole spheroids in a 50 ml conical tube and the media was replaced with 19 ml OM. After addition of the other cell types the contents were mixed well and 200 µL was transferred to each well of a 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate (Thermo Fisher Scientific) to produce 96 spheroids. The media was refreshed every 2 days for 7 days, and then the spheroids were transferred to 6-well Ultra-Low Attachment Microplates (Corning), with no more than 16 spheroids per well. Plates were maintained on a belly dancer shaker (IBI Scientific) at speed of 4.75, and the media was exchanged every 4–7 days.