Imaris 7

Spheroid cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and incubated with antibodies against CD133 (MBS462020; Miltenyi Biotec), N-cadherin (#14215; Cell Signaling), Nanog (#4893, #8822; Cell Signaling Technology) and/or PIK3R3 (sc-376615; Santa Cruz Biotechnology) in a solution of PBS with 1% FBS and 0.1% Triton X-100 at 4 °C overnight. Cells were then stained with secondary antibodies tagged with anti-mouse Alexa Fluor 488 (A-11029; Thermo Fisher), and Alexa Fluor 568 (A-11061; Thermo Fisher). Nuclei were counterstained using DAPI. Stained cells were visualized on an inverted confocal microscope (Leica Microsystems) and images were processed using Imaris 7.6.

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.