Thapsigargin increases autophagy in RASF. OASF and RASF were incubated with thapsigargin (0, 0.1, 0.5, 1 or 5 μM) for 60 h. Autophagic cell number was determined by autophagic vesicles. Data represent means ± S.E. (n = 4) (a). Thapsigargin (5 μM) was added for 0, 12, 24, 36 or 48 h. After incubation, total protein was extracted. SDS-PAGE and immunoblotting were performed with anti-beclin, LC3 or actin antibody (b: upper). The expression of beclin and LC3-II was quantified, compared with the expression of actin (b: lower). OASF and RASF were treated with thapsigargin (5 μM) for 60 h. Crystal violet-stained cells are shown by light microscopy (upper panel) and GFP-LC3-transfected cells are shown by fluorescent microscopy (lower panel) (c). (a) to (b): *P < 0.05, versus non-treated OASF. #P < 0.05, versus OASF with same treatments.