P16 clone jc8

The following antibodies were used for immunoblotting; p16 (clone JC8, sc-56330, Santa Cruz Biotechnology), p14 ARF (Santa Cruz sc-8613), beta Actin (Santa Cruz, sc-69879), Flag (Sigma, F1804), AICDA (Cell signaling, #4975), SMAD3 (Invitrogen, 51–1500), Rb1 (cell signaling, #9309), SETD7 (Cell signaling, #2825), and alpha Tubulin (Sigma, T6074), and chromatin immunoprecipitation; CtBP1 (EpiGentek, #A2705), HDAC1 (Thermo Fisher PA1-860), and p300 (Bethyl A300-358A) and p53 (Sant Cruz, sc126).

Specimens were fixed in 10% formaldehyde and embedded in paraffin. Three-micrometer-thick sections were cut from the paraffin-embedded tissue blocks and stained with hematoxylin and eosin. Paraffin sections of each tissue sample were used for immunohistochemical staining with antibodies to SOX4 (polyclonal antibody, ab86809, dilution 1:60; Abcam, Cambridge, MA, USA) and p16 (clone: JC8, dilution 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Immunohistochemical staining was performed using the automated Bond Max Stainer (Leica Biosystems, Wetzlar, Germany).

Mice were treated in accordance with the guidelines of the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” and the Austrian law. Animal experiments were approved by the ethics committee of the Austrian Federal Ministry of Science and Research (Application No. Zl. 188809/13). Six week old Crl:SHO-Prdkc^scidHr^hr female mice were purchased from Charles River (Germany). Human ASCs were infected with shCntrl or shDIRAS3 expressing lentiviruses. Cells were puromycin selected and 4 days later 1.5 × 10⁵ cells were injected subdermally into the flank of each mice (n=5 per group). Six weeks later, Forane® anesthetized mice were sacrificed and transplanted tissue site was surgically removed, fixed, paraffin embedded, sectioned and stained. Tissue sections were selected based on the hematoxylin and eosin stain. Antigen retrieval was performed by boiling in 6.5mM sodium citrate buffer (pH 6) for 10 min. 3%BSA/PBS was used for blocking. Sections were incubated at 4 °C overnight with anti p16^INK4A antibody (1:50) (p16 (clone JC8), Santa Cruz). Anti-mouse secondary Ab was applied for 1hr at room temperature. DAB chromogen was added for 1 min, washed followed by hematoxylin staining for 1 min and mounting. Number of p16 positive cells was counted per section.