Figure 4. Effects of p97 inhibition on cell death and formation of polyubiquitinated protein aggregates and vacuoles in RASFs under conditions of endoplasmic reticulum stress.
Cells were transfected with siRNA targeting p97 or control siRNA (0.5 μM siRNA to 2.5 × 105 cells) and then treated for 72 hours with 5 μM thapsigargin (TG) in presence or absence of 5 mM 3-methyladenine (3-MA). Dead cells were evaluated microscopically (original magnification x100) and by flow cytometry using annexin V/propidium iodide (PI) staining. Numbers in each compartment are the percentage of cells (A, B). Cells were treated as above (in absence of 3-MA), fixed and stained with anti-ubiquitin antibodies (green) and DAPI (blue). Original magnification ×400 (C). Ubiquitin-positive dots per cell were calculated with a software (D). * = p < 0.05; ** = p < 0.01; *** = p < 0.001.