Fig. 7.

Protective effects of FCPR03 in ameliorating ER stress and reducing apoptosis are abolished by silencing of HO-1. (A, C, E) HT-22 cells were transfected with a negative control (NC) or siRNA of HO-1 (si–HO–1). At 24 h after transfections, cells were pretreated with FCPR03 for 1 h and subjected to 6 h of OGD. The protein levels of HO-1, GRP78, p-eIF2α, and eIF2α were determined by Western blotting. (B, D, F) The relative levels of HO-1/β-tubulin (n = 3), GRP78/β-tubulin (n = 3) and p-eIF2α/eIF2α (n = 3) were measured by semiquantitative analysis of the Western blots. (G, H) HT-22 cells transfected with NC or si–HO–1 were pretreated with FCPR03 for 1 h, subjected to 6 h of OGD, and were incubated with CellROX Deep Red Reagent (5 μM) for 30 min. Finally, the intracellular ROS was detected by confocal microscopy. Intracellular ROS was quantified by Image J (18 fields from three independent experiments, n = 3). (I) HT-22 cells transfected with NC or si–HO–1 were pretreated with FCPR03 for 1 h and subjected to OGD for 6 h and re-oxygenation for 24 h. Then, cell viability was measured by a CCK-8 assay (12 duplications from three independent experiments, n = 3). (J, K) HT-22 cells were processed as described above, and the protein level of cleaved-caspase3 was determined by Western blotting. The relative level of cleaved-caspase 3/pro-caspase 3 was measured by semiquantitative analysis of the Western blots (n = 3). Results are expressed as the mean ± SD, *P < 0.05, **P < 0.01 versus the indicated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)