TRPV1 siRNA assay.

<p>Upper panels: A. TRPV1 siRNA – sequence No. 1: Knockdown of TRPV1, using siRNA, in HEK-293 and HT-29 cells resulted in inhibition of Hsp90, and Hsp70 expression. HEK293 & HT-29 cells were treated with either oligofectamine alone, heat shock at 42°C for 3 hrs, heat shock at 42°C for 3 hrs with the transfection of 100 nM TRPV1 siRNA or with the transfection of 200 nM TRPV1 siRNA. Cells were transfected with TRPV1 siRNA for 72 hrs, prior to heat shock. Lanes grouped by curly brackets represent repeated RNAi experiments. B. TRPV1 siRNA – sequence No. 2 & scrambled siRNA: Knockdown of TRPV1, using a second sequence of siRNA and 200 nM of scrambled siRNA in HEK-293 and HT-29 cells, resulted in inhibition of Hsp90, and Hsp70 expression.Lower panel: Representative immunoblot of TRPV1 abundance. TRPV1 was detected in HEK-293 cells treated with heat shock at 42°C for 3 hrs with the transfection of either 100 nM TRPV1 siRNA or 200 nM TRPV1 siRNA. Lanes grouped by curly brackets represent repeated RNAi experiments.</p

Effect of various TRPV1 agonists and antagonists on heat shock protein expression.

<p>A. Capsaicin & Capsazepine: Representative immunoblots analysis of Hsp70, Hsp90 and Hsp27 abundances. Hsp70, Hsp90 and Hsp27 were detected in MLE-12 (Panel a), HEK-293e (Panel b) cells. Hsp70 was detected in Cells in L3 – primary cells isolated from human lung tumors (Panel d). Cells culture were incubated with 0.01% DMSO, 4 µM, 16 µM or 32 µM of capsaicin (Caps) for 1 hr. 15 µg of total protein/lane were separated on 9% SDS-PAGE gel. (Panel c) represents Hsp70 and Hsp90 levels in control HEK-293e cells, HEK-293e cells treated with 32 µM of capsaicin for 1 hr, heat shock treatment for 3 hrs at 42°C and cells treated with both 32 µM of capsaicin for 1 hr and heat shock for 3 hrs at 42°C.B. RTX & AMG-9810: Representative immunoblot analysis of Hsp70, Hsp90. Hsp70, Hsp90 were detected in MLE-12 and HT-29 cells. Cell cultures were incubated with 0.01% DMSO, 10 nM, 40 nM, 50 nM or 80 nM of RTX for 1 hr, 80 nM RTX with the addition of 5 µM of AMG-9810 for 1 hr, 5 µM AMG-9810 at 37°C for 1 hr or 10–50 µM AMG-9810 at 42°C for 3 hrs. 15 µg of total protein/lane were separated on 9% SDS-PAGE gel.</p

Capsazepine inhibits HSF-1 expression and nuclear translocation.

<p>A. Representative immunoblot analysis of nuclear HSF-1 and phospho-HSF-1 abundances. Intra-nuclear HSF-1 and Phospho-HSF-1 were detected in HEK-293e cells. Cells were incubated with 4 µM, 16 µM or 32 µM of capsaicin for 1 hr. HEK-293e cells were treated as indicated with heat shock at 42°C for 3 hrs, heat shock at 42°C for 3 hrs with the addition of 5 mM EGTA, heat shock at 42°C for 3 hrs with the addition of 100 µM capsazepine or 100 µM capsazepine with the addition of capsaicin. B. Semi-quantitative RT-PCR for HSF-1. Upper panel: mRNA levels from non treated HEK293e cells (control), cells treated with 32 µM capsaicin for 1 hr (Caps), cells treated with heat shock at 42°C for 3 hrs, cells treated with heat shock at 42°C for 3 hrs with the addition of 100 µM capsazepine. Lower Panels: GAPDH mRNA levels from HEK293e cells as indicated above as well as densitometric analysis (Right panel). C. Electrophoretic mobility shift assay for HSF-1 DNA binding activity. Nuclear protein isolated from HEK-293e cells treated with 4 µM, 16 µM or 32 µM of capsaicin for 1 hr. Cells treated as indicated with heat shock at 42°C for 3 hrs, heat shock at 42°C for 3 hrs with the addition of 5 mM EGTA, heat shock at 42°C for 3 hrs with the addition of 100 µM capsazepine or 100 µM capsazepine with the addition of capsaicin. 10 µg of nuclear protein incubated for 20 min at room temperature with a ³²P-labeled double-stranded DNA oligonucleotide containing a consensus –HSF-1 binding site. Right panel indicates densitometric analysis.</p

HEK293e cells versus HEK cells and S2 KO cells – TRPV1 & TRPV4 expressions.

<p>A. Representative immunoblot analysis of Hsp70 and TRPV1 in HEK with or without the transfection of the TRPV1 gene (HEK, HEK+V1) and S2 drosophila cells lacking the TRPV1 gene (S2). B. Semi-quantitative RT-PCR for TRPV4. Upper panel: mRNA levels from HEK and PC3 prostate cancer cells: non treated cells (DM), cells treated with 32 µM capsaicin for 1 hr (Cps) and cells treated with heat shock at 42°C for 3 hrs (HS) or with heat shock at 42°C for 3 hrs with the addition of capsaicin (Cps+HS). Lower panels: mRNA levels from HEK-293e cells: non treated cells (DM), cells treated with 32 µM capsaicin for 1 hr (Cps), cells treated with heat shock at 42°C for 3 hrs (HS), and cells treated with heat shock at 42°C for 3 hrs with the addition of 32 µM (Cps+HS) and mRNA levels of GAPDH.</p

TRPV1 expression in HEK293e cells.

<p>A. Left panel: Double immunofluorescence staining of TRPV1 and Hsp70 in HEK-293e cells treated with 32 µM capsaicin for 1 hr. TRPV1 visualized in green, Hsp70 in red and DAPI (Nuclei) in blue. White arrows indicate Hsp70 abundance (left picture) and co-localization (right picture). B. Right panel: Immunofluorescence staining of Hsp70 in HEK-293e cells in non-treated cells, treated cells with 32 µM of capsaicin for 1 hr, heat shock treated cells for 3 hrs at 42C and cells transfected with 200 nM TRPV1 siRNA and treated with heat shock for 3 hrs at 42C.</p

Capsaicin and capsazepine effects on heat shock protein expression in HEK293e, MLE-12, MCF-7 and HT-29 cells.

<p>Representative immunoblot analysis of Hsp70, Hsp90 and Hsp27 abundances. Hsp70, Hsp90 and Hsp27 were detected in HEK293e (Panel A), MLE-12 cells (Panel B), HT-29 (Panel C) and MCF-7 (Panel D). Cells in culture were incubated with 0.01% DMSO, 32 µM of capsaicin (Caps) for 1 hr, 32 µM of capsaicin (Caps) for 1 hr with the addition of 100 µM capsazepine (CPZ) or 100 µM capsazepine alone at 37°C. Cells were treated with either heat shock at 42°C for 3 hrs, heat shock at 42°C for 3 hrs with the addition of 100 µM capsazepine. Cells were treated with 32 µM of capsaicin with the addition of 5 mM EGTA, 5 mM EGTA alone at 37°C or heat shock at 42°C for 3 hrs with the addition of 5 mM EGTA. 15 µg of total protein/lane were separated on 9% SDS-PAGE gel.</p

TRPV1 mRNA and protein abundances in HEK-293e cells.

<p>A. Semi-quantitative RT-PCR for TRPV1. Upper left panel: mRNA levels from PC3 prostate cancer cells: non treated cells, cells treated with 32 µM capsaicin for 1 hr and cells treated with heat shock at 42°C for 3 hrs. Lower right panel: mRNA levels of GAPDH as well as densitometric. Upper right panel: mRNA levels from HEK-293e cells: non treated cells, cells treated with 32 µM capsaicin for 1 hr (Caps), cells treated with heat shock at 42°C for 3 hrs, and cells treated with heat shock at 42°C for 3 hrs with the addition of 32 µM capsaicin. Lower right Panel: mRNA levels of GAPDH as well as densitometry. B. Representative immunoblot analysis of TRPV1 abundance. TRPV1 was detected in HEK-293e cells. Cells in culture were incubated with 0.01% DMSO or treated with 4 µM, 16 µM or 32 µM of capsaicin for 1 hr. C. Representative immunoblot analysis of TRPV1 and Hsp90 abundances in HEK-293e cells. Cells were treated with 32 µM capsaicin for 0 hr, 1 hr, 4 hrs and 6 hrs.</p

AdHSP alters interactions between Caspase 8, 9, 3 and Apaf-1.

<p>A and B: Representative Autoradiogram Demonstrating AdHSP treatment disrupts interaction between caspase 8 and caspase 9. Representative autoradiograms. 250 µg of cytosolic extracts were immunoprecipitated with rabbit polyclonal antibody to caspase 8 or caspase-9 and subjected to SDS-PAGE. Immunoblotting performed with either primary rabbit polyclonal antibodies to caspase 9 or caspase-8, secondary goat anti rabbit IgG. Lower panels: IgG detection. IgG serves as loading control. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026956#pone-0026956-g001" target="_blank">Figure 1</a>. <b>C: Hsp70 disrupts caspases 3, 8 9 and Apaf-1 complexes.</b> 250 µg of cytosolic extracts from lung tissue fractionated via column chromatography, eluted by molecular weight, immunoprecipitated with an antibody to caspase-9 and subjected to 9% SDS-PAGE. Molecular weight of each fraction (kDa) indicated at the top of the figure. Detecting antibodies (anti-caspase 9, anti-caspase 8, anti-pro-caspase 3, anti-Hsp70 and anti-Apaf-1,) noted to the left of the panels. Lower panel: IgG detection. IgG serves as loading control. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026956#pone-0026956-g001" target="_blank">Figure 1</a>.</p

AdHSP reduces Myeloperoxidase (MPO) in 2CLP-induced lung injury.

<p>A. Myeloperoxidase (MPO) immunostaining. 20× Magnification. MPO containing cells stain brown. <b>Left panel</b>: 2CLPAdHSP, <b>Right panel</b>: 2CLPPBS. TUNEL and MPO double staining. 40× Magnification. <b>Left panel</b>: Myeloperoxidase (MPO) Immunostaining, MPO containing cells stain brown. <b>Right panel</b>: TUNEL fluorescent staining. TUNEL positive cells are green. White arrows depict apoptotic neutrophils – brown staining for MPO and green for TUNEL. Yellow arrows depict apoptotic cells that are negative for MPO but positive for TUNEL. Red arrows depict nuclei stained for dapi –showing the orientation of the slide. Black arrows depict MPO-positive cells that were not apoptotic. TUNEL and Aquaporin 5 (AQP5) double staining. AQP5 – a specific marker for alveolar type I cells. 40× magnification. Left panel: AQP5 immunostaining, AQP5 containing cell stain brown. Right panel: TUNEL fluorescent staining. TUNEL positive cells are green. Red arrows indicate positive staining of alveolar type I cells (both AQP5 and TUNEL). Yellow arrows indicate alveolar type II cells that are negatively stained for AQP5 and positively stained for TUNEL.</p

AdHSP prevents nuclear translocation of activated Caspase-3.

<p>A. Representative autoradiograms for pro-caspase 3 and activated (cleaved) Caspase 3. 30 µg of cytosolic (upper panels) and nuclear (lower panel) extracts were subjected to SDS-PAGE. Immunoblotting performed with primary rabbit polyclonal antibody to pro- caspase 3 and secondary goat anti rabbit IgG, primary goat antibody to β-actin and secondary donkey anti goat IgG, primary rabbit polyclonal antibody to active (cleaved) caspase-3 and secondary goat anti rabbit IgG, primary mouse monoclonal antibody to histone (H1) and secondary goat anti mouse IgG.. β-actin and histone serve as loading controls. <b>B. Representative stained fixed tissue section depicting intra-nuclear staining for Caspase 3.</b> Sections obtained from T0 control, 2CLPPBS and 2CLPHSP rats. Tissue isolated 48 hrs after the induction of sepsis. <b>Upper panel</b>: 40× magnifications. Black arrows indicate active caspase 3 stained nuclei. <b>Lower panel</b>: 100× magnification of upper panel. <b>C. Caspase 3 Activity Assay.</b> Graphic representation of relative caspase-3 enzymatic activity (mean +/− standard deviation) * = significantly different from Control and AdHSP+TNF.</p