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: Journal of Extracellular Biology

Article Title: Contamination of bacterial extracellular vesicles (bEVs) in human urinary extracellular vesicles (uEVs) samples and their effects on uEVs study

doi: 10.1002/jex2.69

Figure Lengend Snippet: Bacterial overgrowth in the urine and contamination of bacterial proteins in human nanoscale uEVs samples. (A) and (B): After removal of cells and debris, the urine samples (30 ml/aliquot) with or without 10 mM NaN 3 were immediately (0 h) subjected to turbidity measurement and bacterial colony forming unit (CFU) count, or stored at 25°C for 8 or 24 h before these measurements. (C): The nanoscale uEVs were then isolated from these urine samples and their enrichment was confirmed by Western blot analysis of exosomal markers, including Alix, HSP70 and Rab5. (D): The amounts of total proteins were measured from nanoscale uEVs samples derived from the urine immediately after collection (0 h) or stored at 25°C for 8 or 24 h with or without NaN 3 . All quantitative data were derived from three independent experiments using different biological samples (each with triplicate measurements) and are reported as mean ± SD.

Article Snippet: The membranes were incubated in PBS containing 5% skim milk for 1 h and then with each of mouse monoclonal antibodies against exosomal markers, including Alix (Jeppesen et al., ; Zhao et al., ), HSP70 (Singhto et al., ), and Rab5 (Singhto & Thongboonkerd, ) (all were from Santa Cruz Biotechnology; Santa Cruz, CA), diluted 1:250, 1:1,000 and 1:500, respectively, in PBS containing 1% skim milk at 4°C overnight.

Techniques: Isolation, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Effects of HDAC inhibitors, arimoclomol and combined treatments on HSPA1A in motor neurons expressing TDP-43 G348C . A Phase contrast image of spinal cord – DRG culture. Arrows point to motor neurons. B Small but significant increase in the percentage of motor neurons with HSPA1A immunoreactivity three days following microinjection of plasmid encoding TDP-43 G348C , compared to absence of labeling in neurons injected with mCherry (control). C-F Percentage of HSPA1A immunopositive motor neurons after three days of treatment with vehicle, the HDAC inhibitors C SAHA, D RGFP109, E RGFP963 or F Tubastatin A, or with arimoclomol alone or in combination with the respective HDAC inhibitor. HDAC inhibitors with class I activity ( C-E ) significantly increased the percentage of neurons expressing HSPA1A. Arimoclomol and the HDAC6 inhibitor Tubastatin A were ineffective. Data presented as mean ± S.D., n = 9 cultures. Statistical significance was evaluated by one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 20μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Expressing, Microinjection, Plasmid Preparation, Labeling, Injection, Control, Activity Assay

Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Hspa1a mRNA in ALS culture models using single molecule fluorescence in situ hybridization (smFISH). Cultures containing motor neurons expressing TDP-43 G348C , FUS R521G or SOD1 G93A were treated with vehicle (DMSO), 4 µM arimoclomol, 1µM RGFP963 or the combination of arimoclomol and RGFP963 (Combo). smFISH was conducted on day three. A The highly expressed Hspa8 mRNA is presented as a reference control for comparison to the minimal labeling of Hspa1a mRNA in B,C,D . B No significant impact of TDP-43 G348C or drug treatments on the number of Hspa1a mRNA spots. C Scarcity of Hspa1a mRNA spots in neurons expressing FUS R521G ; no significant effect of drug treatments. D Low numbers of Hspa1a mRNA spots in neurons expressing SOD1 G93A ; a small but significant increase in a subset of neurons by arimoclomol and RGFP963 treatments. Data are presented as mean ± S.D. n = 9-24 neurons per group. E-G Number of transcription sites by smFISH. E Example of H spa1a transcription site labeling. F,G Effect of ALS variants and combination drug treatment on the number of Hspa1a transcription sites on F day one and G day three following microinjection of expression vectors. n = 6-30 neurons per group. Statistical significance was evaluated through one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05. Scale bar = 15μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Fluorescence, In Situ Hybridization, Expressing, Control, Comparison, Labeling, Microinjection