MPNST724 and ST88-14 human MPNST cell lines were obtained from Jonathan A. Fletcher laboratory at DFCI and have been tested mycoplasma free. MPNST 724 was grown in RPMI with 10% FBS and ST88-14 was grown in RPMI with 15% FBS. cDNA for wild-type human EED and SUZ12 in pDONR vectors were obtained from Harvard PlasmidID and cloned into MSCV-based retroviral vector with FLAG-HA (FH) tag (Addgene plasmid 41033)43 (link) using Gateway technology. To generate stably expressing cell lines, MPNST724 and ST88-14 were infected with empty vector, MSCV-FH-EED, MSCV-FH-SUZ12 and selected using puromycin (2 μg/ml for 72 hours). Growth curve of the infected cells was performed using Alamar blue cell viability reagent (Life Technology, DAL1100).
For immunofluorescence of infected cell lines, cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton X-100, and blocked for 1 hours using 10% goat serum. The cells were then incubated for 2 hours in primary antibody (H3K27me3, #9733, Cell Signaling, 1:400) followed by secondary antibody (Alexa-594 conjugated goat-anti-rabbit, Invitrogen). Slides were mounted using Prolong Gold with DAPI (Invitrogen). Photographs were taken on a Nikon microscopy using a Roeper Scientific camera.
For immunofluorescence of infected cell lines, cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton X-100, and blocked for 1 hours using 10% goat serum. The cells were then incubated for 2 hours in primary antibody (H3K27me3, #9733, Cell Signaling, 1:400) followed by secondary antibody (Alexa-594 conjugated goat-anti-rabbit, Invitrogen). Slides were mounted using Prolong Gold with DAPI (Invitrogen). Photographs were taken on a Nikon microscopy using a Roeper Scientific camera.
