FIGURE 5.

Pharmacological and genetic inhibition of the E1 enzyme does not affect nucleolar stress-induced PICT1 degradation in H1299 cells. A, H1299 cells were treated with 1 μm epoxomicin (Epoxo), 10 μg/ml Pyr41, or vehicle (DMSO) for 5 min, followed by the addition of 1 mm FUrd, 5 nm ActD, DMSO, or no addition (−). After incubation for 6 h, cell lysates were prepared and subjected to immunoblot analysis as described under “Experimental Procedures.” B–D, HeLa cells were transfected with indicated siRNAs using Lipofectamine RNAi Max according to the manufacturer's protocol. B, synthesized siRNAs with target sequence. C, after a 72-h incubation, RNAs were extracted, and the expression of UBE1, UBA6, and ACTB mRNAs was analyzed by RT-PCR. Primers used are represented. D, cells were also treated for the indicated times with 5 ng/ml ActD. Cell lysates were then subjected to immunoblot analysis as described under “Experimental Procedures.”