Wild type fvb n mice

An SMA mouse model carrying two SMN2 copies on one allele and a murine Smn null, FVB/N background was used [35 (link)]. It was established by breeding Smn^KO/KO; SMN2^tg/tg mice with Smn^KO/WT mice resulting in 50% SMA mice (Smn^KO/KO; SMN2^tg/0) and 50% phenotypically normal heterozygous littermates (Smn^KO/WT; SMN2^tg/0) [36 (link)]. FVB/N wild type mice were used as controls (Charles River).

Male/female FVB/N wild-type mice (Charles River) aged 10 and 90 days were sacrificed, and tissues were surgically removed, frozen in dry ice and stored in a freezer at −80°C until analysis. Before protein, RNA and DNA extractions, tissues were pulverized in liquid nitrogen and divided into three homogeneous aliquots. Animal procedures were approved by the ICGEB Animal Ethics committee. This committee works according to the Italian law D.L.vo n.26/2014, which establishes and regulates animal experimentation.

Wild-type FVB/N mice were obtained from Charles River (Wilmington, MA). Hydrodynamic injection was performed as described [41 (link)]. In brief, 10μg pT3-EF5α-PIK3CAH1047R (with a HA tag) and pT3-EF5α-YapS127A (with a Flag tag) along with sleeping beauty transposase (SB) in a ratio of 25:1 were diluted in 2 ml saline (0.9% NaCl), filtered through 0.22 μm filter, and injected into the lateral tail vein of 6 to 8-week-old FVB/N mice in 5 to 7 seconds. Mice were housed, fed, and monitored in accordance with protocols approved by the Committee for Animal Research at the University of California, San Francisco.

Wild-type FVB/N mice were obtained from Charles River Laboratories (Wilmington, MA, USA) and the Rictor^fl/fl mice^{29 (link)} from the Jackson Laboratory (Sacramento, CA, USA). Hydrodynamic injection was performed as described previously.^{30 (link)} In brief, the plasmids encoding the genes of interest along with SB transposase in a ratio of 25:1 were diluted in 2 ml saline (0.9% NaCl), filtered through 0.22 μm filter, and injected into the lateral tail vein of the mice in 5–7 s. For the tumorigenesis models, 20 μg sgPten, 20 μg c-Met with 1.6 μg SB plasmid were delivered into FVB/N mouse liver (n=14). To determine the requirement of mTORC2 in sgPten/c-Met-driven liver carcinogenesis, high dose of Cre (60 μg) or pT3-EF1α (60 μg) was mixed with sgPten (20 μg), c-Met (20 μg) and SB (4 μg) and injected into Rictor^fl/fl mice (n=11 and 5, respectively). Mice were housed, fed and monitored in accordance with protocols approved by the Committee for Animal Research at the University of California, San Francisco.

Wild-type FVB/N mice purchased from Charles River (Tranent, Scotland) were used throughout this study (incorporating both sexes). All animal procedures and breeding were performed in accordance with UK Home Office guidelines (PPL P92BB9F93) and were approved by an internal ethics committee at the University of Edinburgh.