Immunofluorescence

ML-1 cells (n = 1.5 × 10⁵) were seeded on poly-d-lysine–coated, 12-mm glass coverslips in 24-well plates and allowed to attach for 24 hours at 37°C. Cells were then treated with vehicle or with the indicated drugs (see Results) at the indicated experimental settings and fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS for 15 minutes at RT. Cells were then permeabilized by 0.5% Triton X-100 (Thermo Fisher Scientific) in PBS for 10 minutes at RT, and blocked for 1 hour at RT with 1% normal goat serum (Cell Signaling) in PBS. After the blocking step, cells were incubated overnight at 4°C with the primary antibodies diluted in blocking buffer. Indirect immunofluorescence was performed using rabbit monoclonal anti phospho-eIF2α (1:100; Cell Signaling), mouse monoclonal anti-CHOP (CCAAT/enhancer-binding protein homologous protein; 1:500; Cell Signaling). Secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (1:500; Thermo Scientific) were used for 1 hour at RT. We added 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific) for nuclear stain. After final washes with PBS, the coverslips were mounted on microscope slides with Vectashield mounting media (Vector Laboratories). The stained slides were observed with a Leica DM6000 microscope.