Supplemental Material for Matsumoto and Akashi, 2018

supplemental figures and table

Data of Behavioral sensitivity to heat and threshold temperature of TRPA1

The file includes three sheets:(1) Data_behav_exp_Tbody, body temperature that elicits the first attempt at escaping from heat for three Anolis species; (2)Data_bheav_exp_Tplate,hot plate temperature that elicits the first attempt at escaping from heat for three Anolis species; (3)Data_TRPA1_threshold, arrhenius break temperature (ABT) as an index of temperature threshold of TRPA1for three Anolis specie

HSF1 and HSF3 cooperatively regulate the heat shock response in lizards

<div><p>Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of <i>HSP</i> expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog <i>HSF</i> genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.</p></div

Heat shock induces the HSE-binding activity of HSF1 and HSF3 in lizard and frog cells.

<p>(<b>A</b>) Expression of HSF1 and HSF3 in mouse (MEF), chicken (DF-1), lizard (GL-1), and frog (Speedy) cells. MEF and DF-1 cells maintained at 37^°C were heat shocked for 1 h at 42^°C and 45^°C, respectively. GL-1 cells maintained at 30 and Speedy cells maintained at 28^°C were heat shocked for 1 h at 42 and 37^°C, respectively. Cell extracts (48 μg protein per sample) were subjected to western blotting using anti-HSF1 (anti-cHSF1x), anti-HSF3 (anti-XtHSF3-2), anti-HSF2 (anti-mHSF2-4), or anti-β-actin antibodies. Arrows indicate specific bands of HSF1 in MEF (70 kDa), DF-1 (72 and 65 kDa), GL-1 (72 kDa), and Speedy (72 kDa) cells, those of HSF2 in MEF (70 kDa), DF-1 (80 kDa), GL-1 (74 kDa), and Speedy (68 kDa) cells, and those of HSF3 in DF-1 (75 kDa), GL-1 (74 kDa), and Speedy (76 kDa) cells in unstressed conditions. Stars indicate non-specific bands. (<b>B</b>) Induction of the HSE-binding activity in mouse, chicken, lizard, and frog cells during heat shock. Cells were heat shocked as described in A. Whole cell extracts were prepared and aliquots (10 μg proteins) were subjected to EMSA in the presence of 2 μl of 1:10-diluted preimmune (PI) or anti-HSF2 (anti-mHSF2-4) serum. (<b>C</b>) Analysis of heat-induced HSE-binding activity. Whole cell extracts from heat-shocked cells described in B (10 μg proteins) were subjected to antibody supershift experiments using anti-HSF1 (anti-cHSF1γ) or anti-HSF3 (anti-mHSF3-1, anti-cHSF3γ, anti-AsHSF3-1, or anti-XtHSF3-2) antibodies in the presence of anti-HSF2 (anti-mHSF2-4) antibody. (<b>D</b>) Gel filtration analysis of HSF1 and HSF3. Whole cell extracts described in B were subjected to gel filtration [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref024" target="_blank">24</a>]. Proteins were subjected to western blotting using anti-HSF1 (anti-cHSF1x) or anti-HSF3 (anti-XtHSF3-2) antibodies. The elution positions of monomers, trimers, and dimers are shown at the bottom. (<b>E</b>) Oligomeric states of HSF1 and HSF3 in lizard and frogs. HSF1 stayed mostly as an inert monomer in unstressed condition, whereas HSF3 was an inert dimer. Upon heat shock, both HSF1 and HSF3 converted to active trimers that bind to the HSE.</p

Lizard and frog HSF3 as well as HSF1 can induce HSP70 expression.

<p>(<b>A</b>) Induction of HSP70 by lizard HSFs. HSF1-null MEF cells (HSF1-/-) were infected for 48 h with adenovirus expressing GFP, AsHSF1-HA, AsHSF2-HA, AsHSF3-HA, or AsHSF4-HA. These cells and wild-type MEF cells (HSF1+/+) were untreated (C) or treated with heat shock at 42^°C for 1 h and allowed to recover for 3 h (HS + R). Cell extracts were prepared from these cells and aliquots were subjected to western blotting using the indicated antibodies. Positions of molecular weight markers are indicated. Only the lower band of AsHSF3-HA (arrow) was detected by anti-HA antibody. (<b>B</b>) Induction of HSP70 by frog HSFs. HSF1-null cells were infected with adenovirus expressing GFP, XtHSF1-HA, XtHSF2-HA, XtHSF3-HA, or XtHSF4-HA. These cells were treated and analyzed as described in A. (<b>C</b>) Induction of HSP70 by vertebrate HSF1 members. HSF1-null cells were infected with adenovirus expressing GFP, XtHSF1-HA, AsHSF1-HA, cHSF1, or hHSF1, and were treated as described in A. Aliquots of cell extracts were subjected to western blotting using the indicated antibodies including anti-HSF1 (anti-cHSF1x). (<b>D</b>) Induction of HSP70 by vertebrate HSF3 members. HSF1-null cells were infected with adenovirus expressing GFP, XtHSF3-HA, AsHSF3-HA, cHSF3, or mHSF3, and were treated as described in A. Aliquots of cell extracts were subjected to western blotting using the indicated antibodies including anti-HSF3 (anti-XtHSF3-2 or anti-mHSF3-1). (<b>E</b>) Induction of HSP70 mRNA by vertebrate HSF1 and HSF3 members. HSF1-null cells (HSF1-/-) were infected with the indicated adenoviruses for 48 h. These cells and wild-type MEF cells (HSF1+/+) were untreated (C) or treated with heat shock at 42^°C for 30 min (HS). HSP70 mRNA levels were quantified by RT-qPCR are showen (n = 3). *, p < 0.01 versus each control value; **, p < 0.05 versus control value of GFP-expressing cells by Student’s t-test.</p

HSF1 and HSF3 induce heat shock response in lizard cells.

<p>(<b>A</b>) Induction of HSP70 in HSF3 knockdown cells. <i>Gekko gecko</i> GL-1 cells were infected with Ad-sh-SCR, Ad-sh-GgeHSF3-KD1, or Ad-sh-GgeHSF3-KD2 for 96 h, and were then heat shocked at 42^°C for 1 h and allowed to recover for 3 h (HS + R3) or 6 h (HS + R6). Cell extracts were prepared from these cells, and aliquots were subjected to western blotting using anti-HSP70 (anti-cHSP70a), anti-HSF3 and anti-β-actin antibodies. (<b>B</b>) Induction of HSP70 in HSF3 knockout cells. Wild-type (WT) and HSF3-null GL-1 cells (HSF3-/- clones 303 and 109) were heat-shocked at 42^°C for 1 h and allowed to recover for 3 h (HS + R3) or 6 h (HS + R6). Cell extracts were prepared and aliquots were subjected to western blotting. (<b>C</b>) Induction of HSP70 in HSF1 knockdown cells. GL-1 cells were infected with Ad-sh-SCR, Ad-sh-GgeHSF1-KD1, or Ad-sh-GgeHSF1-KD2 for 96 h, and were treated as in A. (<b>D</b>) Induction of HSP70 in HSF3 and HSF1 knockdown cells. GL-1 cells were infected with the indicated adenoviruses for 96 h, and were treated as in A. Cell extracts were prepared from these cells, and aliquots were subjected to western blotting using antibodies including anti-HSP70, anti-HSP40 (anti-hHSP40-1), and anti-HSP60 (anti-mHSP60-1) antibodies. (<b>E</b>) Cell survival under heat shock conditions. GL-1 cells were infected with Ad-sh-SCR, Ad-sh-GgeHSF1-KD1, Ad-sh-GgeHSF3-KD1, or both viruses for 96 h, and were then heat shocked at 45^°C for the indicated periods. These cells were stained with trypan blue, and percentages of surviving cells are shown (n = 3). *, p < 0.05; **, p < 0.01 by ANOVA.</p

Diagrammatic representation of vertebrate HSF family members.

<p>The percent identity between XtHSF1 and each HSF was established. The number of amino acids of each HSF is shown at the amino-terminal end. DBD, DNA-binding domain; HR, hydrophobic heptad repeat; DHR, downstream of HR-C. The red box in mHSF3 indicates an HR-C-like domain, in which hydrophobic amino acids are not well conserved. AsHSF and XtHSF members were identified in this study. cHSF1, cHSF2, and cHSF3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref021" target="_blank">21</a>]; cHSF4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref014" target="_blank">14</a>]; mHSF1 and mHSF2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref048" target="_blank">48</a>]; mHSF3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref014" target="_blank">14</a>]; mHSF4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref038" target="_blank">38</a>]; hHSF1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref039" target="_blank">39</a>]; hHSF2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref040" target="_blank">40</a>]; hHSF4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180776#pone.0180776.ref041" target="_blank">41</a>].</p