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. 2014 Sep 9;33(20):2374–2387. doi: 10.15252/embj.201488648

Figure 4. Endogenous or exogenous overexpression of HSF1 ameliorates the negative effects of aneuploidy on protein folding.

Figure 4

A, B Western blot analysis of HSF1 and HSP90 expression in HCT116 8/3* c1-c4 (A). Loading control: GAPDH. Shown are representative images of at least 3 independent experiments. Quantification of the signal intensities from the Western blots (B), calculated relative to control cells (which were set to 1).

C FlucDM-mCherry was expressed in parental HCT116* and HCT116* 8/3 c1-c4 for 36 h. Cells were then incubated with either solvent control (DMSO) or 50 nM 17-AAG for 8 h followed by measurement of luminescent activity. The depicted values show the percentage of luminescence in cells treated with 17-AAG relative to DMSO-treated cells (which were set to 100%).

D Parental HCT116* and HCT116* cells trisomic for chromosome 8 (HCT116 8/3* c1-c4) were treated with the indicated concentrations of 17-AAG, and cell number was determined 72 h thereafter. Cell number is shown as the percentage of the DMSO-treated control (which was set to 100%).

E Western blot analysis of HSF1 expression and its downstream targets in the indicated aneuploid cells transfected with ca-HSF1. Loading control: GAPDH. Shown are representative images of at least 3 independent experiments. Quantification of the signal intensities normalized to the loading control is shown above the images.

F RPE-1* 21/3 cells were transiently transfected with either pCDNA or ca-HSF1 by electroporation. Forty-eight hours post-transfection cells were incubated with the indicated concentrations of 17-AAG, and cell number was determined 72 h thereafter. Cell number is shown as the percentage of the DMSO-treated control.

G FlucDM-mCherry was co-expressed with either pCDNA or ca-HSF1 in the indicated cell lines for 36 h. Cells were then incubated with either solvent control (DMSO), 50 nM 17-AAG (HCT116), or 5 nM 17-AAG (RPE-1) for 8 h followed by measurement of luminescent activity. The depicted values show the percentage of luminescence in cells treated with 17-AAG relative to DMSO-treated cells (which were set to 100%).

H Western blot analysis of HSF1 expression and its downstream targets in the indicated aneuploid cells transfected with ca-HSF1 using electroporation. Loading control: GAPDH. Shown are representative images of at least 3 independent experiments. Quantification of the signal intensities normalized to the loading control is shown above the images.

I Control HCT116* cells were transiently transfected with either pCDNA or ca-HSF1 by electroporation. Fourty-eight hours post-transfection cells were incubated with the indicated concentrations of 17-AAG and cell number was determined 72 h thereafter. Cell number is shown as the percentage of the DMSO-treated control.

J Control RPE-1* cells were transiently transfected with either pCDNA or ca-HSF1 by electroporation. Fourty-eight hours post-transfection cells were incubated with the indicated concentrations of 17-AAG and cell number was determined 72 h thereafter. Cell number is shown as the percentage of the DMSO-treated control.

Data information: The data are the mean of at least three independent experiments ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; non-parametric t-test.