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. 2018 Aug 9;293(38):14758–14774. doi: 10.1074/jbc.RA118.002913

Figure 2.

Figure 2.

Overexpression of Hsp70 reduces the aggregation and cell death induced by mutant PKCγ. A, representative images of cells expressing V138E PKCγ–GFP (green) and empty vector, Hsp70 (red), or Hsp70–T13G (red) in CHO cells. Scale bar = 10 μm. B and C, CHO (B) and SH-SY5Y (C) cells expressing WT or V138E PKCγ–GFP and Hsp70 or Hsp70–T13G were quantified to obtain the percentage of cells containing aggregates. One to 2 days after transfection, the cells were fixed, permeabilized, and incubated with anti-Hsp70 antibody. At least 300 GFP- and Hsp70-positive cells were counted. Mean ± S.E. of the mean, n = 2–3. *, p < 0.05; **, p < 0.01; and ***, p < 0.001; one-way analysis of variance with Tukey's post hoc tests. D, representative immunoblots with anti-Hsp70, Hsc70, Hsp40, Hsp27, and α-tubulin antibodies using CHO and SH-SY5Y cell lysates. E, quantification of the results shown in D. n = 4. **, p < 0.01; one-way analysis of variance with Tukey's post hoc tests. F, representative immunoblots with anti-PKCγ, Hsp70, Hsc70, and α-tubulin antibodies using CHO and SH-SY5Y cell lysates with or without V138E PKCγ–GFP expression. The cells were assessed 1 day after transfection. G, SH-SY5Y cells expressing WT, R41P, Q127R, or F643L PKCγ–GFP and Hsp70–K71E were quantified to obtain the percentage of cells containing aggregates. One day after transfection, the cells were fixed, permeabilized, and incubated with anti-Hsp70 antibody. At least 200 GFP- and Hsp70-positive cells were counted. Mean ± S.E. of the mean, n = 3. *, p < 0.05, and **, p < 0.01; Student's t test. H, percentages of cell death in high GFP-positive cells. Two days after transfection, the CHO cells were harvested and assessed by flow cytometry. Mean ± S.E. of the mean, n = 3. *, p < 0.05, and **, p < 0.01; Student's t test. I, mean percentages of cell death in high GFP-positive cells. Two days after transfection, the CHO cells were harvested and assessed by flow cytometry. Mean ± S.E. of the mean, n = 3. *, p < 0.05; **, p < 0.01; and ***, p < 0.001; one-way analysis of variance with Tukey's post hoc tests.