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. 2011 Aug;31(15):3146–3157. doi: 10.1128/MCB.01187-10

Fig. 2.

Fig. 2.

HspB1 abolishes toxicity of Aβ oligomers. (A) Dose-response curves for cytotoxicity of the Aβ conformers—unassembled (Unas) and oligomers (Oligo)—on N2a cells. Cell viability was monitored by CellTiter Blue assays. The x axis corresponds to monomer concentrations of Aβ. (B) Titration of HspB1 against Aβ oligomers. Cell viability values at various concentrations of monomeric HspB1 were plotted as the percentage of toxicity inhibited. Toxicity of Aβ oligomers (10 μM as monomer) in the absence of HspB1 was set to zero, and cell viability in buffer control was set to 100%. The values were fitted to a ligand binding equation with an R2 of 0.86. (C) N2a cells were treated with 10 μM Aβ conformers—unassembled (Unas), oligomers (Oligo), or fibrils (Fiber)—before (light gray bars) or after (dark gray bars) coincubation with purified human HspB1 (2 μM as monomer). Cells treated with buffer or HspB1 alone (None) had no influence on cell viability. (D) Effect of the order of addition of HspB1 on oligomer-mediated toxicity. The concentrations of all components were identical to those in panel B. The various treatments are numbered 1 to 5, and the treatments are listed in steps 1 to 3. Cells treated with buffer (treatment 1), oligomer (treatment 2), or oligomer coincubated with HspB1 (treatment 3) confirmed the results described in panel B. In treatment 4, cells were first treated with HspB1, followed by the addition of oligomer without washing off the HspB1. In treatment 5, cells were first treated with HspB1, which was washed away prior to oligomer addition. (E) Accumulation of reactive oxygen species (ROS) in N2a cells was detected using DCFDA fluorescence by microscopy. The left panels show differential interference contrast (DIC) images, and the right panels show fluorescence images (ROS). Incubation of N2a cells with Aβ oligomers (10 μM as monomer) alone resulted in the accumulation of ROS in nearly all cells. Treatment of cells with buffer or HspB1 (2 μM as monomer) alone did not induce ROS. Cells treated with oligomers preincubated with HspB1 did not accumulate ROS. (F) Quantitation of data shown in panel E by ImageJ software.