Cd 1 male nude nu nu mice

CD-1 male nude (nu/nu) mice, 6–8 weeks old and weighing 22-24 g were purchased from Charles River Laboratories (Calco, Italy). The procedures involving mice were in compliance with Regina Elena National Cancer Institute animal care guidelines and with national and international directives (D.L. March 4, 2014, no. 26; directive 2010/63/EU of the European parliament and of the council; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 2011).
For the intracardiac experimental metastasis model, nude mice (age 8–10 weeks) were anesthetized and injected with 2×10⁶ cells of NRAS^Q61L (2/14, 2/17) or BRAF^V600E (2/21, 2/33) clones, suspended in 100 μl sterile PBS, into the left ventricle of the heart by nonsurgical means. The spontaneous, pulsatile entrance of bright red oxygenated blood into the transparent needle hub indicated proper positioning of the needle. After 5 weeks, the mice were sacrificed; selected organs were excised from the mice at necropsy and were preserved in 10% formalin solution for subsequent scoring of metastasis. NRAS^Q61L (2/14, 2/17) and NRAS/Sema6A cells (2/14, and 2/17) were injected intracardially as above described, and after 7 weeks the mice were sacrificed and the organs were processed and analyzed as described above. Box plot of in vivo metastasis analysis was obtained by software Prism version 6.0.

Six- to eight-week-old CD-1 male nude (nu/nu) mice weighing 22–24 g were purchased from Charles River Laboratories (Calco, Italy). The procedures involving mouse care were in compliance with the Regina Elena National Cancer Institute animal care guidelines and with international directives (directive 2010/63/EU of the European parliament and council; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 2011). To evaluate the tumorigenic ability of LMNA-KD cells or sphere-derived adherent cells, the mice were injected subcutaneously into the left flank with various concentrations of cells (from 5 × 10⁴ to 1 × 10⁷ cells/mouse) in 200 μl of Matrigel (BD Biosciences-Discovery Labware). Each group included five mice. Tumor sizes were measured three times a week in two dimensions using a caliper, and tumor weight was calculated using the following formula: a × b²/2, where a and b are the long and short diameters of the tumor, respectively.