Figure 8: HSF1–SSBP1 complex supports cell survival and mitochondrial function.

(a) HSF1–SSBP1 complex protects cells from heat shock. MEF cells (HSF1+/+, clone #10) were infected with Ad-sh-mHSF1-KD2 or Ad-sh-SCR for 24 h, and then infected with adenovirus expressing wild-type hHSF1, each hHSF1 mutant or GFP for 48 h. After incubating the cells at 45 °C for 2 h, the number of viable cells excluding trypan blue was counted (left) and thiazolyl blue tetrazolium bromide (MTT) assay was performed (right; n=3). Mean±s.d. is shown. Asterisks indicate P<0.05 by Student’s t-test. (b) HSF1–SSBP1 complex protects cells from a proteasome inhibitor. MEF cells were treated as described in a, and were incubated with MG132 (10 μM) for 6 h. The number of viable cells was counted (left) and MTT assay was performed (right; n=3). (c) Heat shock and proteasome inhibition reduce mitochondrial membrane potential. MEF cells were infected with Ad-sh-SCR, Ad-sh-mHSF1-KD2 or Ad-sh-mSSBP1-KD1 for 72 h. After being incubated at 42 °C for 1 h or with MG132 for 6 h, the cells were stained for 30 min at 37 °C with DAPI (blue) and a fluorescent dye TMRM (red) that accumulates in the mitochondria in a manner dependent on membrane potential. FCCP (10 μM) was added for 10 min as a negative control. Scale bars, 20 μm. Representative fluorescence images are shown, and TMRM fluorescence intensities (arbitrary units) are compared with that in unstressed scrambled RNA-treated cells were quantified using ImageJ (n=3). Mean±s.d. is shown. Asterisks indicate P<0.05 by Student’s t-test. (d) HSF1–SSBP1 complex supports maintenance of mitochondrial membrane potential in proteotoxic conditions. Cells were infected with Ad-sh-mHSF1-KD2 or Ad-sh-SCR for 24 h, and then infected with adenovirus expressing wild-type hHSF1, each hHSF1 mutant or LacZ for 48 h. After being incubated at 42 °C for 1 h or with MG132 for 6 h, the cells were stained with TMRM in the absence or presence of FCCP as described in c. Scale bars, 20 μm. TMRM fluorescence intensities (arbitrary units) compared with that in scrambled RNA-treated cells (white bars) were quantified using ImageJ (n=3).