Matrigel layer

The effects of miR-877-3p silencing on CC cell invasion were also determined. To do this, C-33A, SiHa and HeLa cells were transfected with NC and anti-miR-877-3p, as described above, for 4 days. Cells were FBS-starved overnight, trypsinized and 1.25 × 10⁵ cells were re-seeded in FBS-free DMEM on a 40-µL Matrigel^® layer (BD, Franklin Lakes, NJ, USA), which had been previously allowed to gel for 15–30 min at 37 °C in Transwell^® inserts with an 8-µm-pore membrane (Sarstedt, Nümbrecht, Germany). Inserts were placed in wells with 10% FBS-containing medium, and maintained for three more days. Finally, the gel layer was removed with a cotton-tipped swab, and invading cells that had reached the membrane were fixed and stained with a paraformaldehyde-containing crystal violet solution. Images were captured under a Leica DMi1 microscope with the Leica Application Suite v4.12 program (Leica, Wetzlar, Germany) at 50× magnification. The area occupied by invading cells was measured with the NIS Elements program (Nikon Instruments, Amsterdam, Netherlands).

Migration assays were performed in transwell inserts with 8-μm-pore membrane filters, while invasion assays were performed with 8-μm transwell inserts pre-coated with a growth factor-reduced Matrigel layer to mimic basement membranes (BD Biosciences). BCC cells were cultured alone or co-cultured with control lnASCs, leptin shRNA lnASCs, control obASCs, or leptin shRNA obASCs for 7 days in a 1:1 ratio. BCCs were purified with FACS, and 1.25 × 10⁴ BCCs suspended in 50 μl were added to the apical chamber. A total of 200 μl of chemoattractant (10 % FBS; Atlanta Biologicals) was added to the basal chamber and incubated for 4 hours or 24 hours for migration or invasion, respectively. After the allotted time, the lower side of the transwell insert was carefully washed with cold PBS and non-migrating or non-invading cells remaining on the top chamber were removed with a cotton tip applicator. Migrating and invading cells were stained with Calcein-AM (2 μg/ml; Invitrogen) and measured on a fluorescent plate reader (FLUOstar optima, BMG Labtech Inc., Durham, NC, USA). Data were normalized to the respective BCCs without previous exposure to ASCs.

The invasion and migration assays were performed in 24-well Millicell hanging inserts (Millipore) with or without a Matrigel layer (BD Biosciences) according to the manufacturer's instructions. Briefly, 1 × 10⁵ cells were seeded into the top chamber, and DMEM with 10 % FBS was added to the bottom chamber as a chemoattractant. After a 48 hour incubation at 37 °C, the numbers of cells that invaded the Matrigel (invasion) or membrane (migration) were counted in 10 fields using a 40× objective lens.