Ab 105 c

1 x 10⁵ 7940b tumor cells were mixed with 5 x 10⁵ fibroblasts, resuspended in a 50:50 mix of serum-free media [DMEM+1% Pen/Step] and Matrigel (Corning 354234). Two fibroblast clones (in equal numbers) were used in each injection to reduce the impact of clonal variability. Cells were injected subcutaneously into the flanks of NU/J mice (Jackson Laboratory Stock No: 002019). Tumors were measured every other day with calipers, and animals were sacrificed after 10 days. For NK cell depletion experiments, 10μl of anti-asialo GM1 (Wako 986–10001) or an equivalent volume of normal Rabbit IgG control (R&D AB-105-C) was diluted 1:10 in sterile PBS and injected intraperitoneally. Injections were given 24h before tumor implantation, on the day of tumor implantation, and once every three days for the remainder of the experiment.

Migration assays were carried out in a 24-well transwell system equipped with porous (8 μm) polycarbonate membranes. 2 × 10⁵ MB cells were resuspended in 600 μL of growth medium supplemented with 1% FBS and plated into the lower chamber of the transwell system; 5.0 × 10⁴ BMSC cells were resuspended in 200 mkl of growth medium supplemented with 1% FBS and plated into Transwell inserts which were then placed into another transwell system with the lower chamber filled with 600 μl of serum-free medium. After 24 hours the cells on the inserts or in lower chambers were transfected as indicated, 6 hours after the transfection the media was changed and the inserts with BMSC, were placed into the wells with MB cells and incubated at 37 °C for 24 h. When indicated, CXCR4 (Abcam, #ab10403) or SDF1 (CXCL12) (Abcam, #ab9797) antibodies were added to the medium at 10 mkg/ml and 4 mkg/ml concentrations respectively. The inserts were then discarded, and upper sides of the filters were swabbed to remove the cells that did not cross the membrane. The cells present on the lower side of the filters were then fixed in 4 % paraformaldehyde, stained with DAPI and the counted under the microscope. All the experiments were performed in duplicate. The following control antibodies were used: rabbit IgG control (AB-105-C, R&D systems) and mouse IgG2b isotype control (MAB004, R&D systems).

Cells were washed with PBS, fixed in 4% PFA for 15 min, and permeabilized for 10 min in 0.5% [v/v] Triton X-100 (Sigma Aldrich). After blocking in 1% bovine serum albumin (BSA), fixed and permeabilized SMCs were incubated with anti-human alpha-smooth muscle actin (α-SMA; 1:200, Dako, M0851), TOM20 (1:200, Proteintech, 11802-1-AP), alpha-tubulin (α-tubulin, 1:25, Cell Signaling, 2144S), calponin (1:50, Thermo Scientific, MA5-32061), mouse IgG control (DAKO, X0931), or rabbit IgG control (R&D systems, AB-105-C). Subsequently, after washing Alexa Fluor 594 (1:1000, Thermo Scientific, R37115) or 488 (1:1000, Life Technologies, A32723) labeled secondary antibody was applied. Nuclei were counterstained with 2.5 μg/ml 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Carl Roth), and slides were covered using a mounting medium (Dako).
For mitochondrial visualization, cells were labeled with 300 nM MitoTrackerRed FM (ThermoFisher Scientific) in serum-free CM at 37°C for 30 min. Nuclear staining was performed with 1 μM Hoechst 33,342 solution (Thermo Scientific). Images were acquired using a Leica DMI6000B inverted fluorescence microscope.