Journal: Journal of Sport and Health Science

Article Title: Exercise attenuates stress-related signaling as sensed by higher phosphorylation of small heat shock proteins in skeletal muscle from older individuals

doi: 10.1016/j.jshs.2025.101111

Figure Lengend Snippet: Relative HSP abundances in whole skeletal muscle homogenates from young adults and older adults pre and post HIT exercise. Representative Westen blots of (A) HSP72, HSP27, and αB-crystallin and (B) phosphorylated HSP27 Ser15 (pHSP27 Ser15) and pαB-crystallin Ser59 in whole muscle homogenates from the vastus lateralis of the same individuals. Calibration curves of mixed muscle homogenates are indicated and were used to determine the relative number of given proteins (see Methods). Stain-free gels are indicative of total protein loading, and molecular weights are indicated by markers collected under white light capture without moving the membrane between that and chemiluminescence detection. Relative abundances of (C) HSP72, (D) HSP27, (E) pHSP27 Ser15, (F) αB-crystallin, and (G) pαB-crystallin Ser59 from young (circle) and older adults Pre (square) and older adults Post (triangle) HIT exercise are shown relative to average Old (pre) on a given gel (data are presented as mean ± SD). Individuals indicated by the number of symbols ( n : 5–7), with the same color assigned to the same individual and consistent across all graphs. * p ≤ 0.05 indicates Brown-Forsye and Welch’s and post hoc analysis using Games-Horwell. HIT = high-intensity training; HSP = heat shock protein; pαB-crystallin Ser59 = phospho-αB-crystallin at Serine59; pHSP27 Ser15 = phospho-HSP27 at Serine15.

Article Snippet: Details of antibodies used are as follows: HSP72 (1 in 500 mouse monoclonal, SMC100A; StressMarq Biosciences, Victoria, Canada); HSP27 (1 in 1000 mouse monoclonal, G3.1 ab2790; Abcam, Cambridge, UK); pHSP27 Ser15 (1 in 2000 monoclonal rabbit, ab76313; Abcam), pHSP27 Ser82 (1 in 2000 polyclonal mouse, ADI-SPA-524; Enzo Biochem, Farmingdale, NY, USA), αB-crystallin (1 in 1000 mouse monoclonal, SPA-222; StressGen Biotechnologies), pαB-crystallin Ser59 (1 in 1000 rabbit polyclonal, SPA-227; StressGen Biotechnologies).

Techniques: Staining, Membrane

Journal: Journal of Sport and Health Science

Article Title: Exercise attenuates stress-related signaling as sensed by higher phosphorylation of small heat shock proteins in skeletal muscle from older individuals

doi: 10.1016/j.jshs.2025.101111

Figure Lengend Snippet: HSP abundances in type I and II skeletal muscle fibers from young and older adults. (A, C, and F) The MHC isoform present was determined in individual muscle fiber segments from the vastus lateralis and, following pooling into type I and type II groups from a given biopsy, were analyzed by Westen blotting. Westen blots of (A) HSP72, (C) HSP27 and pHSP27 Ser15, (F) αB-crystallin and pαB-crystallin Ser59, with MHC isoforms in groups of fibers. Stain-free gels are indicative of total protein loading, and molecular weights are indicated by markers collected under white light capture without moving the membrane between that and chemiluminescence detection. Calibration curves of mixed muscle homogenates are indicated. Relative protein abundances of (B) HSP72, (D) HSP27, (E) pHSP27 Ser15, (G) αB-crystallin, and (H) pαB-crystallin Ser59 in fibers from young (circle) and older adults (square) type I fibers (no outline) and type II fibers (outline). All fibers are expressed relative to the average older adult’s type I fibers. The same color is assigned to the same individual and is consistent with (data are presented as mean ± SD). * p < 0.05 and ** p < 0.01, mixed effect model Univariant using either Tukey’s or Games-Horwell’s multiple comparison test (see Methods). HIT = high-intensity training; HSP = heat shock protein; MHC = myosin heavy chain; pαB-crystallin Ser59 = phospho-αB-crystallin at Serine59; pHSP27 Ser15 = phospho-HSP27 at Serine15.

Article Snippet: Details of antibodies used are as follows: HSP72 (1 in 500 mouse monoclonal, SMC100A; StressMarq Biosciences, Victoria, Canada); HSP27 (1 in 1000 mouse monoclonal, G3.1 ab2790; Abcam, Cambridge, UK); pHSP27 Ser15 (1 in 2000 monoclonal rabbit, ab76313; Abcam), pHSP27 Ser82 (1 in 2000 polyclonal mouse, ADI-SPA-524; Enzo Biochem, Farmingdale, NY, USA), αB-crystallin (1 in 1000 mouse monoclonal, SPA-222; StressGen Biotechnologies), pαB-crystallin Ser59 (1 in 1000 rabbit polyclonal, SPA-227; StressGen Biotechnologies).

Techniques: Staining, Membrane, Comparison

Journal: Journal of Sport and Health Science

Article Title: Exercise attenuates stress-related signaling as sensed by higher phosphorylation of small heat shock proteins in skeletal muscle from older individuals

doi: 10.1016/j.jshs.2025.101111

Figure Lengend Snippet: HSP abundances in type I and II skeletal fibers from older adults pre- and post HIT exercise. Relative protein abundances of (A and B) HSP72, (C and D) HSP27, (E and F) pHSP27 Ser15, (G and H) αB-crystallin, and (I and J) pαB-crystallin Ser59 in fibers from old pre and old post HIT exercise. All fibers are expressed relative to the average old pre type I fibers or relative pre type II depending on fiber type. The same color is assigned to the same Individual, consistent in both graphs and all figures. * p < 0.05 and ** p < 0.01 indicated significant difference in paired t -test (except pHSP27 Ser15 Wilcoxon match-pair rank test). Representative blots are shown in . HIT = high-intensity training; HSP = heat shock protein; pαB-crystallin Ser59 = phospho-αB-crystallin at Serine59; pHSP27 Ser15 = phospho-HSP27 at Serine15.

Article Snippet: Details of antibodies used are as follows: HSP72 (1 in 500 mouse monoclonal, SMC100A; StressMarq Biosciences, Victoria, Canada); HSP27 (1 in 1000 mouse monoclonal, G3.1 ab2790; Abcam, Cambridge, UK); pHSP27 Ser15 (1 in 2000 monoclonal rabbit, ab76313; Abcam), pHSP27 Ser82 (1 in 2000 polyclonal mouse, ADI-SPA-524; Enzo Biochem, Farmingdale, NY, USA), αB-crystallin (1 in 1000 mouse monoclonal, SPA-222; StressGen Biotechnologies), pαB-crystallin Ser59 (1 in 1000 rabbit polyclonal, SPA-227; StressGen Biotechnologies).

Techniques:

Journal: The Journal of Experimental Biology

Article Title: Zebrafish parental progeny investment in response to cycling thermal stress and hypoxia: deposition of heat shock proteins but not cortisol

doi: 10.1242/jeb.244715

Figure Lengend Snippet: Effects of experimental treatments on the gill cellular stress response in adult female zebrafish. (A) Gill hsf1 , hsp70a , hsp90aa and hsp47 relative gene expression. (B) Representative western blot and HSP70, HSP90 and HSP47 relative protein expression. Gene expression data were normalized and expressed as stated in Fig. 2 . Western blot shows HSP70 standard (lane 1), HSP90 standard (lane 2), pool of heat-stressed gills (positive control; lane 3), control treatment (lane 4), cycling temperature treatment (lane 5), cycling hypoxia treatment (lane 6), combined exposure treatment (lane 7) and blank (lane 8). Dashed lines around a lane represent the splicing of separate gel images. Protein expression was normalized to Coomassie stain band intensity and expressed relative to the control treatment for each protein. Values are means+s.e.m. ( hsf1 , n =7–8; hsp70a , hsp90aa and hsp47 , n =6–7; HSP70, HSP90 and HSP47, n =5–6). Statistical differences between gene expression values were determined by Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test ( hsf1 , P <0.001; hsp70a , P =0.363; hsp90aa , P <0.001; hsp47 , P <0.001). HSP70 protein expression was square-root transformed prior to analysis; statistical differences between protein expression values were determined by one-way ANOVA followed by post hoc Holm–Šidák tests (HSP70, P <0.001; HSP90, P =0.027) or a Kruskal–Wallis one-way ANOVA (HSP47, P =0.868). Values for a given parameter that do not share a common letter are significantly different from one another.

Article Snippet: Incubations with primary antibody against HSP47 (1:1000; polyclonal rabbit HSP47/SERPINH1, cat. no. 20R-1310, Fitzgerald Industries International), HSP70 (1:5000; polyclonal rabbit HSP70/HSC70, cat. no. AS05083A, Agrisera, Vännäs, Sweden) and HSP90 (1:2500; monoclonal mouse HSP90, cat. no. SMC-107, StressMarq Biosciences Inc., Victoria, BC, Canada) were carried out overnight at 4°C.

Techniques: Expressing, Western Blot, Positive Control, Staining, Transformation Assay

Journal: The Journal of Experimental Biology

Article Title: Zebrafish parental progeny investment in response to cycling thermal stress and hypoxia: deposition of heat shock proteins but not cortisol

doi: 10.1242/jeb.244715

Figure Lengend Snippet: Effects of experimental treatments on the ovary cellular stress response in adult female zebrafish. (A) Ovary hsf1 , hsp70a , hsp90aa and hsp47 relative gene expression. (B) Representative western blot and HSP70, HSP90 and HSP47 relative protein expression. Gene expression data were normalized and expressed as stated in Fig. 2 . Western blot bands and normalization of protein expression are as stated in Fig. 5 . Values are means+s.e.m. ( hsf1 , n =9–10; hsp70a and hsp90aa , n =9–11; hsp47 , n =7–9; HSP70, HSP90 and HSP47, n =5–6). Statistical differences between gene expression values were determined by Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test ( hsf1 , hsp70a , hsp90aa and hsp47 , P <0.001). HSP90 protein expression was square-root transformed prior to analysis; statistical differences between protein expression values were determined by one-way ANOVA followed by post hoc Holm–Šidák tests (HSP90, P =0.288; HSP47, P =0.019) or a Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (HSP70, P =0.002). Values for a given parameter that do not share a common letter are significantly different from one another.

Article Snippet: Incubations with primary antibody against HSP47 (1:1000; polyclonal rabbit HSP47/SERPINH1, cat. no. 20R-1310, Fitzgerald Industries International), HSP70 (1:5000; polyclonal rabbit HSP70/HSC70, cat. no. AS05083A, Agrisera, Vännäs, Sweden) and HSP90 (1:2500; monoclonal mouse HSP90, cat. no. SMC-107, StressMarq Biosciences Inc., Victoria, BC, Canada) were carried out overnight at 4°C.

Techniques: Expressing, Western Blot, Transformation Assay

Journal: The Journal of Experimental Biology

Article Title: Zebrafish parental progeny investment in response to cycling thermal stress and hypoxia: deposition of heat shock proteins but not cortisol

doi: 10.1242/jeb.244715

Figure Lengend Snippet: Effects of parental treatment on zebrafish embryo cellular stress response. (A) hsf1 , hsp70a , hsp90aa and hsp47 relative gene expression, and (B) representative western blot and HSP70, HSP90 and HSP47 relative protein expression in ∼1 hpf embryos derived from adult zebrafish exposed to the experimental treatments. Gene expression data were normalized and expressed as stated in Fig. 2 . Western blot bands and normalization of protein expression are as stated in Fig. 5 . Values are means±s.e.m. ( hsf1 , hsp70a , hsp90aa and hsp47 , n =5–6; HSP70, HSP90 and HSP47, n =5). Statistical differences between gene expression values were determined by Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test ( hsf1 , P =0.014; hsp70a , P <0.001; hsp90aa , P =0.002; hsp47 , P <0.001). HSP70 protein expression was log-transformed prior to analysis; statistical differences between protein expression values were determined by one-way ANOVA followed by post hoc Holm–Šidák tests (HSP70, P =0.010; HSP47, P =0.175) or a Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (HSP90, P =0.004). Values for a given parameter that do not share a common letter are different from one another.

Article Snippet: Incubations with primary antibody against HSP47 (1:1000; polyclonal rabbit HSP47/SERPINH1, cat. no. 20R-1310, Fitzgerald Industries International), HSP70 (1:5000; polyclonal rabbit HSP70/HSC70, cat. no. AS05083A, Agrisera, Vännäs, Sweden) and HSP90 (1:2500; monoclonal mouse HSP90, cat. no. SMC-107, StressMarq Biosciences Inc., Victoria, BC, Canada) were carried out overnight at 4°C.

Techniques: Expressing, Western Blot, Derivative Assay, Transformation Assay

Journal: Science advances

Article Title: Mechanical manipulation of cancer cell tumorigenicity via heat shock protein signaling.

doi: 10.1126/sciadv.adg9593

Figure Lengend Snippet: Fig. 3. Hsp70 plays a critical role in cancer cell growth and survival under mechanical stress in the stiff hydrogel. (A) Heatmap of heat shock protein (Hsp)−asso- ciated gene expression determined by RNA sequencing (RNA-seq) comparing cancer cells in the soft, intermediate, and stiff hydrogels (N = 3). The unit for the color scale was the z score of the log2 expression data shown. All RNA-seq data were normalized to the tissue culture plate (TCP) group. Fluorescent images of staining against (B) Hsp70 (green) from day 1 to 3 and (F) heat shock factors (HSF-1; green) on day 5 with F-actin (red) and nuclei (blue) in the encapsulated CRCs as well as the fluorescence intensity measurement of Hsp70 on day 3 and nuclear localization ratio of HSF-1 on day 5, respectively (n = 60 colonies from N = 3 independent hydrogels, each point representing one colony). Scale bars, 20 μm [(B) and (F)]. (C) Quantification of Hsp70 protein (left) and gene (right) expression by cancer cells in different groups over 72 hours (N = 4, each point representing an independent hydrogel). (D) Schematic illustration of uniaxial compression on cancer cells encapsulated in intermediate stiffness hydrogel and images of fluorescence staining against Hsp70 in cancer cells with or without small interfering RNA (siRNA) knockdown of Hsp70 (siRNA-Hsp70) under varying levels of compression on day 3. Scale bars, 25 μm. (E) Apoptosis rates of CRCs cultured in the intermediate stiffness hydrogel with or without siRNA-Hsp70 under various magnitudes of uniaxial compression on day 3 (N = 4 independent samples). Data represent the means ± SD. Significant difference P values: *P < 0.05 and ***P < 0.001 (ANOVA).

Article Snippet: The equivalent amount of the protein of each sample was separated by 8 to 15% SDS–polyacrylamide gel electrophoresis (Beyotime); blocked with 5% fat-free milk powder (Aladdin) for 2 hours at room temperature; and incubated with primary antibodies to Hsp70, Hsp90 (Santa Cruz Biotechnology, cat. no. sc-101494, RRID:AB_1124018), HSF1, Oct3/4 (Santa Cruz Biotechnology, cat. no. sc-5279, RRID:AB_628051), Sox2 (Santa Cruz Biotechnology, cat. no. sc-365823, RRID:AB_10842165), STAT3, pSTAT3 (Santa Cruz Biotechnology, cat. no. sc-8059, RRID:AB_628292), PI3K (rabbit; Cell Signaling Technology, cat. no. 4292, RRID:AB_329869), pPI3K (rabbit; Cell Signaling Technology, cat. no. 4228, RRID:AB_659940), Akt (rabbit; Cell Signaling Technology, cat. no. 9272, RRID:AB_329827), pAkt (Tyr326; rabbit; Cell Signaling Technology, cat. no. 2968, RRID:AB_1264114), GADPH (all mouse, 1:1000; Santa Cruz Biotechnology), and Nanog (rat; 1:1000; Abcam).

Techniques: Gene Expression, RNA Sequencing, Expressing, Staining, Fluorescence, Small Interfering RNA, Knockdown, Cell Culture

Journal: Science advances

Article Title: Mechanical manipulation of cancer cell tumorigenicity via heat shock protein signaling.

doi: 10.1126/sciadv.adg9593

Figure Lengend Snippet: Fig. 5. The elevated expression of Hsp70 due to mechanical stress enhances cancer cell stemness in the stiff hydrogel. (A) Schematic illustration of the Hsp70/ Hsp90 association assisting phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). (B) Images of immunofluorescence staining against STAT3 (red) and nuclei (blue) in CRCs cultured under various conditions. (C) The Western blot data of Hsp70, Hsp90, STAT3, and pSTAT3 in different groups. (D) Quantification of the nuclear localization of STAT3 in (B). (n = 60, each point represents one nucleus from N = 3 independent hydrogels). (E) Schematic illustration of the transcriptional regulation of Nanog expression by pSTAT3 and its pharmacological blockade by Stattic. (F) Images of immunofluorescence staining against Nanog (green), F-actin (red), and nuclei (blue) in CRCs cultured under various conditions. (G) The Western blot data and (H) relative gene expression of Nanog, sex-determining region Y- box2 (Sox2), and octamer-binding transcription factor 3/4 (Oct3/4) in different groups. (N = 4, each point represents an independent hydrogel). All experiments were assessed on day 5 of culture. Stress relief was performed from day 4 to 5. Data represent the means ± SD. Significant difference P values: *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA).

Article Snippet: The equivalent amount of the protein of each sample was separated by 8 to 15% SDS–polyacrylamide gel electrophoresis (Beyotime); blocked with 5% fat-free milk powder (Aladdin) for 2 hours at room temperature; and incubated with primary antibodies to Hsp70, Hsp90 (Santa Cruz Biotechnology, cat. no. sc-101494, RRID:AB_1124018), HSF1, Oct3/4 (Santa Cruz Biotechnology, cat. no. sc-5279, RRID:AB_628051), Sox2 (Santa Cruz Biotechnology, cat. no. sc-365823, RRID:AB_10842165), STAT3, pSTAT3 (Santa Cruz Biotechnology, cat. no. sc-8059, RRID:AB_628292), PI3K (rabbit; Cell Signaling Technology, cat. no. 4292, RRID:AB_329869), pPI3K (rabbit; Cell Signaling Technology, cat. no. 4228, RRID:AB_659940), Akt (rabbit; Cell Signaling Technology, cat. no. 9272, RRID:AB_329827), pAkt (Tyr326; rabbit; Cell Signaling Technology, cat. no. 2968, RRID:AB_1264114), GADPH (all mouse, 1:1000; Santa Cruz Biotechnology), and Nanog (rat; 1:1000; Abcam).

Techniques: Expressing, Phospho-proteomics, Immunofluorescence, Staining, Cell Culture, Western Blot, Gene Expression, Binding Assay

Journal: Science advances

Article Title: Mechanical manipulation of cancer cell tumorigenicity via heat shock protein signaling.

doi: 10.1126/sciadv.adg9593

Figure Lengend Snippet: Fig. 6. Hsp70 is the key mechanosensi- tive mediator contributing to the en- hanced in vivo tumorigenic and metastatic potential of CRCs primed with mechanical stress in the stiff hy- drogel. (A) Volcano plots for differentially expressed genes (DEGs). Gray, non-DEGs; red, up-regulated DEGs; blue, down-reg- ulated DEGs. FDR, false discovery rate. (B) Bubble chart of oncology-related Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways of DEGs in different groups. The size of the bubbles represents the rich ratio, denoted by the number below the bubble. (C) Schematic illustration of CRCs pretreated in hydro- gels over 5 days and subsequent isolation for subcutaneous or tail vein inoculation into nude mice for in situ–grown tumor model creation over 20 days or for meta- static lung tumor model creation over 2 months with survival time examination, respectively (N = 6 independent mice). Drugs [JG98 and/or doxorubicin (DOX) at 10 mg/kg] or phosphate-buffered saline (PBS) were injected via the tail vein on day 10 after inoculation in both models. (D) Individual growth curves of tumors in animals inoculated by groups (a) to (f), corresponding to each treatment or culture condition. (E) Statistical analysis of end point (day 20) tumor volumes (N = 6 independent mice). (F) Survival curves for each treatment arm and in different groups [N = 6 independent mice, log-rank (Mantel-Cox) test]. (G) Hematoxylin and eosin staining of histological sections of the in situ–grown tumor on Day 20 and lung tumor models on day 30 in different groups. Scale bars, 100 μm. (H) Quantifi- cation of the relative gene expression of Nanog in tissues harvested from the in situ–grown tumors on day 20 (N = 6 in- dependent mice). Data represent the means ± SD. Significant difference P values: *P < 0.05; **P < 0.01; ***P < 0.001. n.s. denotes no significant difference (P > 0.05) (ANOVA).

Article Snippet: The equivalent amount of the protein of each sample was separated by 8 to 15% SDS–polyacrylamide gel electrophoresis (Beyotime); blocked with 5% fat-free milk powder (Aladdin) for 2 hours at room temperature; and incubated with primary antibodies to Hsp70, Hsp90 (Santa Cruz Biotechnology, cat. no. sc-101494, RRID:AB_1124018), HSF1, Oct3/4 (Santa Cruz Biotechnology, cat. no. sc-5279, RRID:AB_628051), Sox2 (Santa Cruz Biotechnology, cat. no. sc-365823, RRID:AB_10842165), STAT3, pSTAT3 (Santa Cruz Biotechnology, cat. no. sc-8059, RRID:AB_628292), PI3K (rabbit; Cell Signaling Technology, cat. no. 4292, RRID:AB_329869), pPI3K (rabbit; Cell Signaling Technology, cat. no. 4228, RRID:AB_659940), Akt (rabbit; Cell Signaling Technology, cat. no. 9272, RRID:AB_329827), pAkt (Tyr326; rabbit; Cell Signaling Technology, cat. no. 2968, RRID:AB_1264114), GADPH (all mouse, 1:1000; Santa Cruz Biotechnology), and Nanog (rat; 1:1000; Abcam).

Techniques: In Vivo, Isolation, In Situ, Saline, Injection, Staining, Gene Expression

Journal: Science advances

Article Title: Mechanical manipulation of cancer cell tumorigenicity via heat shock protein signaling.

doi: 10.1126/sciadv.adg9593

Figure Lengend Snippet: Fig. 7. The proposed schematic summary of the confining stress–activated Hsp70 signaling to promote the survival, proliferation, and stemness of the cancer cells encapsulated in the stiff hydrogel. (A) The stiff hydrogel imposes high confining stress on the encapsulated cells, leading to the activation of the stretch-activated channel TRPV4 and the associated PI3k/Akt signaling. The activation enhances the phosphorylation and nuclear localization of HSF1, which promotes the expression of Hsp70 to resist anoikis and apoptosis under mechanical stress. Subsequently, the abundant cytosolic Hsp70 associates with Hsp90 to bridge the nuclear localization of STAT3 through enhanced pSTAT3 that promotes the transcription of Nanog and enhances cancer malignancy. The mechanoprimed cancer cells exhibit (B) high tumor- igenicity and (C) high metastatic potential after subcutaneous or intravenous inoculation, respectively.

Article Snippet: The equivalent amount of the protein of each sample was separated by 8 to 15% SDS–polyacrylamide gel electrophoresis (Beyotime); blocked with 5% fat-free milk powder (Aladdin) for 2 hours at room temperature; and incubated with primary antibodies to Hsp70, Hsp90 (Santa Cruz Biotechnology, cat. no. sc-101494, RRID:AB_1124018), HSF1, Oct3/4 (Santa Cruz Biotechnology, cat. no. sc-5279, RRID:AB_628051), Sox2 (Santa Cruz Biotechnology, cat. no. sc-365823, RRID:AB_10842165), STAT3, pSTAT3 (Santa Cruz Biotechnology, cat. no. sc-8059, RRID:AB_628292), PI3K (rabbit; Cell Signaling Technology, cat. no. 4292, RRID:AB_329869), pPI3K (rabbit; Cell Signaling Technology, cat. no. 4228, RRID:AB_659940), Akt (rabbit; Cell Signaling Technology, cat. no. 9272, RRID:AB_329827), pAkt (Tyr326; rabbit; Cell Signaling Technology, cat. no. 2968, RRID:AB_1264114), GADPH (all mouse, 1:1000; Santa Cruz Biotechnology), and Nanog (rat; 1:1000; Abcam).

Techniques: Activation Assay, Phospho-proteomics, Expressing