Fig. 3

Inhibition of Atf4 and Xbp1s ER stress arms impairs bortezomib-induced osteogenic differentiation. Alizarin red staining shows the effects of GSK2606414 (a) or MKC3946 (b) on bortezomib-induced osteogenic differentiation in mMSCs (upper panel) and MC3T3-E1 (lower panel) cells. Eighty to 90% confluent MSCs or MC3T3-E1 cells in 35-mm dishes were treated without or with bortezomib (1 nM) in the absence or presence of the PERK inhibitor GSK2606414 (2.5 nM) or IRE1α inhibitor MKC3946 (10 nM) for 8 days. The culture medium was refreshed every 2 days. The images were taken on a Nikon Eclipse TS100 microscope (scale bars represent 50 μM). c, d Western blotting analysis of the effects of GSK2606414 or MKC3946 on bortezomib-induced osteogenic differentiation-related markers in mMSCs (left panel) and MC3T3-E1 cells (right panel). Confluent MSCs or MC3T3-E1 cells were treated without or with bortezomib (1 nM) in the absence or presence of the PERK inhibitor GSK2606414 (2.5 nM) or IRE1α inhibitor MKC3946 (10 nM) for 24 h. Data shown are representative of 3 independent experiments