A549 xenograft model in nude mice

A549-WT and KI clones 1 and 2 (obtained from two independent nucleotransfection rounds) were thawed and kept in culture for two passages in DMEM 10% FCS 1% L-Glutamine. Cells with 50% Matrigel (Corning) were s.c. injected in the right flank of 6–11 nude mice per group (specified in the legend of Fig. ​Fig.3)3) at 2Mi cells per inoculation. Tumor growth was measured bi-weekly for 36–50 days using a caliper. After termination, tumors were collected, weighed, placed into 10% v/v neutral phosphate buffered formalin solution (Avantor) for 24 h for histology prior to tissue processing or snap-frozen in liquid nitrogen for further analysis. Formalin-fixed tumors were rinsed for 5 min under running tap water, dehydrated with graded ethanol concentrations (50% to 100%), cleared with xylene and infiltrated with paraffin overnight using a vacuum infiltration tissue processor (Leica Biosystems). Tumor tissues were then embedded in paraffin blocks. Paraffin sections were stained with haematoxylin and eosin (H&E). Staining for Ki67 was performed on a Ventana Discovery XT immunostainer (Roche Diagnostics), staining for msCD31 and SMA on a BondRX immunostainer (Leica Biosystems). Slides were digitalized using a ScanScope XT slide scanner (Leica Biosystems) with objective x20.