Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML)
Fig 3
CGs do not induce PML expression or association with nuclear matrix.
A, HeLa cells were dispensed into 384-well pates at a density of 2000 cells/wells. The next day the cells were incubated with 2 μM gitoxigenin, 200 nM ouabain and 16 u IFNϒ for 18h. RNA was isolated and RT-qPCR was performed for PML and GUSB. PML expression was normalized to GUSB internal control. Error bars are SEMs of 6 replicates. B, HEK293T were transfected with Flag-PML IV. The next day the cells were treated as indicated for 24h, followed by lysis in RIPA buffer and centrifugation. RIPA supernatants were immunoprecipitated using anti-Flag antibody. The total lysates before immunoprecipitation, the immunoprecipitated complexes and the RIPA-insoluble pellets were separated on SDS-PAGE followed by immunoblotting with anti-Flag (PML) or anti-ACTIN antibody.