12 well plates

Cell viability, apoptosis and ROS were measured by Muse cell analyzer (EMD Millipore Corporation, US). SAEC were cultured in 12-well plates (Celltreat, Shirley, MA, US) and treated with DEPs using staggered starting times as mentioned above. Samples were centrifuged at 1000 × g for 5 minutes (Thermo Scientific CL2 centrifuge) and mixed with Muse agents (life science of Merck KGaA, Darmstadt, Germany) before testing by the analyzer (protocol provided by EMD Millipore Corporation). Each sample had three replicates. One-way ANOVA was performed via Origin 2018 to examine results from the study groups with P < 0.05 set as indicating a statistically significant difference. A series of t-tests (Origin 2018) were conducted to statistically analyze the difference between control (0 h DEP) and other time points in DEP group.

HT1080 parental and resistant cells were plated at 5000 cells/well in 12 well plates (Cell Treat). The following day cells were treated with either DMSO (Sigma) or with KPT-185 (0, 3.7, 12.3, 111, 333, or 1000 nM for generation of resistance, or 1 μM to evaluate resistance). On days 0, 4, 6, and 8 cells were fixed and stained with Gentian Violet (RICCA Chemical Company) and imaged with a digital camera (Sony Cybershot).

One hundred nanograms of FLAG-A3G-pcDNA or HA-A3G-pcDNA variants were cotransfected with 200 ng of either Vif-pcDNA or pcDNA3.1(+) empty vector into human embryonic kidney 293T cells (American Type Culture Collection) in 12-well plates (CELLTREAT) using the X-tremeGENE 9 DNA Transfection Reagent (Roche). Cells were incubated at 37°C in 5.0% CO₂. At 24 hours after transfection, 2 μM N-carbobenzyloxy-l-leucyl-l-leucyl-l-leucinal (Sigma-Aldrich) or dimethyl sulfoxide control was added. At 48 hours after transfection, the cells were washed once with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer with 1× cOmplete Protease Inhibitor (Roche). The lysates were then subjected to Western blot with anti-FLAG M2 monoclonal antibody (mAb) from mouse (Sigma-Aldrich), anti–α-tubulin mAb from mouse (GeneTex), and anti-Vif mAb from mouse (National Institutes of Health AIDS Reagent Program #319) as primary antibodies. Cy3-labeled goat anti-mouse mAb (GE Healthcare) was used as a secondary antibody to detect the signal. The fluorescent signal was detected and visualized with Typhoon RGB Biomolecular Imager (GE Healthcare).