Research Paper Volume 14, Issue 20 pp 8221—8242

Figure 14. Over-expression of GRP78 suppresses the drug-induced expression of BAG3. (A) HCN2 cells were transfected with a scrambled siRNA or with siRNA molecules to knock down the expression of GRP78, HSP70 or HSP90. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunostaining performed to determine the expression of BAG3 and ERK2. (n = 3 +/-SD) # p < 0.05 greater than vehicle control; † p < 0.05 less than corresponding value in CMV cells. (B) HCN2 cells were transfected with an empty vector plasmid or with plasmids to over-express GRP78, HSP70 or HSP90. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunostaining performed to determine the expression of BAG3 and ERK2. (n = 3 +/-SD) # p < 0.05 greater than vehicle control; † p < 0.05 less than corresponding value in CMV cells. (C) HCN2 cells were transfected with a scrambled siRNA or an siRNA to knock down BAG3 expression. In parallel, they were transfected with a plasmid to express LC3-GFP-RFP. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 4h and 8h. The mean number of intense GFP+RFP+ and RFP+ punctae per cell were determined (n = 3 +/-SD) * p < 0.05 less than corresponding siSCR value; # p < 0.05 greater than corresponding value at 4h. (D) HCN2 cells were transfected with a scrambled siRNA or with an siRNA to knock down BAG3 expression. In parallel, they were transfected with plasmids to express either Tau or APP. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunostaining performed to determine the expression of Tau, APP and ERK2. (n = 3 +/-SD) * p < 0.05 less than vehicle control; ** p < 0.05 less than corresponding AR12 value.