Figure 4

NIM811 induced a transient upregulation of UPR and autophagy. (a and b) In all, 0 μM NIM811 transiently induced UPR signaling (P-EIF2a, Bip, ATF4, CHOP upregulation) at 6 h, but except for Bip, the activation of UPR mediators disappeared at 9 h. Tunicamycin (Tm) and thapsigargin (Tg) were used as positive controls for the UPR. (c) In total, 10 μM NIM811 was added to GFP-LC3B transfected U251 cells and imaged by fluorescence microscopy at 0 h and 6 h. Scale bar=25 μm. LC3 puncta were detected at 3–6 h. LC3 puncta formation occurred before the appearance of vacuoles. (d) LC3-I and -II conversion occurred at 4–6 h of 10 μM NIM811 treatment accompanied by p62 degradation. (e and f) In all, 10 μM NIM811 incubation caused p62 accumulation at 24 h, which persisted at later time points (40–46 h). (g) Immunofluorescence staining of SQSTM1/p62 (red) and nucleus (blue) after 24 h vehicle (left) or 10 μM NIM811-treated (right) U251 cells. Scale bar=25 μm