For microscopy, SF11435 cells were grown on glass coverslips and transduced with either wild-type SETD2-EGFP or SETD2K2R-EGFP using polybrene at 70% confluency. Forty-eight hours post transfection, cells were fixed in 4% PFA for 8 minutes, blocked in 2.5% FBS, 200mM glycine, and 0.1% Triton X-100 in PBS for 30 minutes, incubated with anti-GFPandanti-H3K36me3 (Abcam, Cambridge, UK) primary antibodies overnight at 4°C, washed, and incubated with Alexa Fluor secondary antibodies (Thermo Fischer Scientific, Waltham, MA) for 1 hour at room temperature. Hoechst 3342 (Life Technologies) was added to the secondary antibody inclubation to mark DNA. Following a final wash, cells were mounted in ProLong Diamond Antifade Mountant (Thermo Fischer Scientific), and immunofluorescence images were collected using a Zeiss 780 confocal microscope system (Carl Zeiss AG, Oberkochen, Germany).
For cell proliferation assays, SF11435 cells were grown in a 96-well plate and transduced with either wild-type SETD2-EGFP or SETD2K2R-EGFP using polybrene at 50% confluency. Forty-eight hours post-transfection, cell proliferation was assayed using the CellTiter 96 Non-Radioactive Cell Proliferation Assay and a GloMax Discovery plate reader per the manufacturer’s protocols (Promega, Madison, WI).
For cell proliferation assays, SF11435 cells were grown in a 96-well plate and transduced with either wild-type SETD2-EGFP or SETD2K2R-EGFP using polybrene at 50% confluency. Forty-eight hours post-transfection, cell proliferation was assayed using the CellTiter 96 Non-Radioactive Cell Proliferation Assay and a GloMax Discovery plate reader per the manufacturer’s protocols (Promega, Madison, WI).