E1 inhibition increases short half-life proteins and induces ER stress. (A) K562 cells were infected with an E1 shRNA lentiviral vector or control sequences and selected as described in Figure 2. Total cell lysates were prepared and analyzed by SDS-PAGE immunoblotting with anti-E1, anti-GRP78, anti-cyclin D3, and anti–α-tubulin antibodies. (B) K562 cells were treated with PYZD-4409 or PYZD_mut (50μM) for 4 hours. After incubation, total cellular proteins were isolated and analyzed by SDS-PAGE immunoblotting with anti-cyclin D3 and anti–α-tubulin antibodies. (C) HCT116 cells were treated with PYZD-4409 or PYZD_mut (50μM) for 2 hours. After incubation, total cellular proteins were isolated and analyzed by SDS-PAGE immunoblotting with anti-p53 and anti–α-tubulin antibodies. (D) K562 cells were treated with PYZD-4409 at the concentrations indicated for 24 hours, and total cellular RNA was isolated. GRP78 and HSP70 mRNA expression was measured relative to 18S RNA by real-time RT-PCR. Data points represent the mean ± SD fold increase of GRP78 and HSP70/18S expression relative to controls (^ΔΔCT normalization). (E) K562 cells were treated with PYZD-4409 (50μM), PYZD_mut (50μM), or DMSO for 2.5 hours. After incubation, total cellular proteins were isolated and analyzed by SDS-PAGE immunoblotting with anti–phospho-JNK, phospho-p38 mitogen-activated protein kinase, and anti-E1. The E1 serves as a protein loading control. (F) MDAY-D2 cells were treated with PYZD-4409 (10μM) or DMSO for 24 hours. After incubation, total cellular proteins were isolated and analyzed by SDS-PAGE immunoblotting with anti–phospho-PERK, anti-CHOP, anti–ATF-4 and antitubulin antibodies.