Drug triggers immunecells to attack prostatecancer WUSTLmedschool NatureComms
treated PBMCs, the PDTOs or C4-2B spheroids were treated with dispase II , followed by incubation for 60 min at 37 °C to digest the Matrigel. PDTOs and C4-2B spheroids were washed twice with ice-cold basal advDMEM/F12 media. To the pellet, 10 µl of binding buffer with propidium iodide and Hoechst was added and incubated for 10 min at room temperature in the dark. The entire content was transferred to a microscopic slide, and fluorescent images were taken in EVOS M5000 imaging system .
For immunoprecipitation studies, cell or organ tissue were lysed by sonication in RLB, the lysates were quantitated and 0.5 to 1 mg of protein lysate was immunoprecipitated using 2 μg of anti-CSK, anti-pTyr, anti-FLAG, anti-HA, anti-Myc or anti-EZH2 antibody coupled with protein A/G sepharose overnight, followed by washes with RLB and PBS buffers. The beads were boiled in sample buffer and immunoblotting was performed as described above.
Inhibition of pY18-CSK upon ACK1 knockdown or pharmacological inhibition was assessed by flow cytometry. Briefly, Jurkat cells treated with vehicle or (
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