Nod scid il2rγ nsg mice

Animal experiments were conducted according to relevant ethical regulations for the use of laboratory animals and after acquired permission from the University of Salamanca Committee for animal experimentation (ref # 0000061). BALB/c-Rag2^nullIL2rγ^null (BRG) mice // or NOD-scid IL2rγ^null (NSG) mice were bred and maintained in the SPF area of the University of Salamanca Animal Facility with controlled environment conditions (20–23 °C, 12:12 light/dark cycles, 30–70% relative humidity) and fed ad libitum. CM-272 was solubilized in 0.9% saline solution. RPMI-8226-luc cells (8 ×10⁶) were injected intravenously into 8-week-old NOD-SCID-IL-2Rγ^−/− (NSG) mice (Charles River Laboratories) and tumor development was monitored by noninvasive bioluminescence imaging (BLI) with a Xenogen IVIS 50 system (Caliper Life Sciences). After 4 weeks, animals were randomized into two groups (n = 6/group) receiving vehicle (0.9% saline solution) or CM-272 (5 mg/kg, 5 times/week by intraperitoneal injection).

A375M2 cells were transfected with either control siRNA or siROCK1/2 (60 nM total for both). Three days later, cells were labeled with 10 μM CMFDA-Green (C7025, Life Technologies) for 10 min and then they were trypsinized and counted. 1x10⁶ labeled cells/0.1ml PBS were injected into tail vein of NOD/SCID/ IL2Rγ−/− mice (NSG, Charles River). 24 h later, TRITC-dextran (70 kDa, D1818, ThermoFisher) was intravenously injected and 10 min later mice were sacrificed. Lungs were extracted, washed with PBS (with calcium/magnesium) twice and fixed with 4% formaldehyde for 16 h at 4°C. Lungs were examined under a confocal microscope (see Immunofluorescence section). Data are presented as % field of area covered by fluorescence (green for cells, red for dextran which represents permeability), n = 5 mice/condition for each experiment, n = 2 independent experiments.

For experimental metastasis assays, A375M2 cells pre-treated in vitro for 24 h with either DMSO or Compound C (2 μM) were labelled with 10 μM CMFDA-Green (C7025, Life Technologies) for 10 min and then trypsinized and counted. 1 × 10⁶ labelled cells / 0.2 ml PBS along with drugs (same concentration as pre-treatment) were injected into tail vein of 9–10 week old female NOD/SCID/ IL2Rγ-/- mice (NSG, Charles River). Mice were sacrificed 30 min (to confirm that equal numbers arrived at the lung) and 24 h after tail vein injection. Lungs were extracted, washed with PBS (with calcium/magnesium) twice and fixed with 4% formaldehyde for 16 h at 4 °C. Lungs were examined under a Zeiss LSM 710 Meta confocal microscope (Carl Zeiss) with a 20X objective. Data are presented as percentage of field of area covered by fluorescence, and 15 fields per mouse were analysed. n = 7 mice/condition for each experiment (2 mice sacrificed 30 min and 5 mice 24 h after tail vein injection), n = 2 independent experiments.