P akt1 2

A standard Western blot analysis of whole-cell protein lysates was performed using primary antibodies against cleaved PARP, PARP (Cell Signaling Technology, #9542, 1:1,000, RRID:AB_2160739), cleaved caspase-3 (Cell Signaling Technology Cat# 9661, 1:1,000, RRID:AB_2341188), caspase-3 (Cell Signaling Technology Cat# 9662, 1:1,000, RRID:AB_331439), cylclinD1 (Cell Signaling Technology Cat# 2978, 1:1,000, RRID:AB_2259616), p27 (Cell Signaling Technology Cat# 3686, 1:1,000, RRID:AB_2077850), p21 (Cell Signaling Technology Cat# 2947, 1:1,000, RRID:AB_823586), pRb (Cell Signaling Technology Cat# 8516, 1:1,000, RRID:AB_11178658), and Rb (Cell Signaling Technology Cat# 9309, 1:1,000, RRID:AB_823629) to check the changes of apoptosis and G0/G1 phase markers. As well, for the alteration of effectors, the signaling pathways were examined with the primary antibodies including p-AKT1/2 (Cell Signaling Technology Cat# 4060, 1:1,000, RRID:AB_2315049), AKT1/2 (Proteintech Cat# 10176-2-AP, 1:2000, RRID:AB_2224574), p-ERK (Cell Signaling Technology Cat# 4370, 1:1,000, RRID:AB_2315112), and ERK (Proteintech Cat# 16443-1-AP, 1:2000, RRID:AB_10603369). Equal amounts of protein, which were blotted with an anti-β-actin antibody (Proteintech Cat# 60008-1-Ig, 1:2000, RRID: AB _2289225), was used as loading control.

Commercially available paraffin-embedded tissue array slides containing 79 human sarcomas and 4 human normal tissues (NBP2-30332; Novus Biologicals, and T242, US Biomax, Inc., USA) were purchased. Sections were deparaffinized, then incubated with antibodies recognizing human CD133 (MBS462020; MyBioSource), Nanog (ab80892; Abcam), and p-Akt1/2 (#9271; Cell Signaling) in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4 °C overnight. Staining was visualized using secondary antibodies tagged with Alexa Fluor 488 (A32766, A10042) and Alexa Fluor 594 (A32744, A32754), from Thermo Fisher, with nuclear counterstaining using DAPI. Images were collected on an inverted confocal microscope (Leica Microsystems) and processed using Imaris 7.6. Five fields were examined for each sample.

Cells were washed with cold PBS and lysed using NP40 (SIGMA-Aldrich, St. Louis, MO, USA). Lysates were centrifuged at 10,000 rpm for 10 min at 4 °C. Proteins were separated on SDS-PAGE polyacrylamide by molecular weight and transferred to PVDF membranes. Transferred membranes were blocked with 5% low-fat milk (Svelty, Nestlé) in TBS-T. Membranes were incubated overnight at 4 °C with primary antibodies p-Akt1/2 and total akt1/2 (Cell Signaling, Danvers, MA, USA) as described previously in [41 (link)].