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: Non-coding RNA Research

Article Title: Exosomal miRNA-218–5p derived from low-passage dermal papilla cells modulates hair follicle growth and development

doi: 10.1016/j.ncrna.2026.01.004

Figure Lengend Snippet: Exosomes derived from low-passage DPCs regulated HFSC proliferation. (A) Indirect immunofluorescence showing ALPL and PCNA expression in low-passage (P1) and high-passage (P8) DPCs (scale bar = 50 μm). (B) RT-qPCR analysis of HF development-related gene expression in P1 and P8 DPCs (unpaired two-tailed t -test, n = 3). (C) TEM images of exosomes from P1 DPCs (DPC-Exos P1) and P8 DPCs (DPC-Exos P8). (D) NTA measurement of particle size of DPC-Exos P1 and DPC-Exos P8. (E) Western blot detection of exosome-specific proteins in DPC-Exos P1 and DPC-Exos P8. (F) CCK-8 assay evaluating HFSC proliferation after treatment with DPC-Exos from P1 and P8 (one-way ANOVA, n = 5). (G) Flow cytometry analysis of HFSC apoptosis after treatment with DPC-Exos from P1 and P8 (one-way ANOVA, n = 3). ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The Annexin V-FITC Apoptosis Detection Kit (Vazyme, China, Cat No. A214) was employed, and apoptosis rates were analyzed via flow cytometry using a FACSAria SORP instrument (Becton Dickinson, USA).

Techniques: Derivative Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Biotechnology Reports

Article Title: Production and characterization of rNGFSP: a recombinant fusion immunogen eliciting dual anti-NGF and anti-Substance P therapeutic antibodies for Degenerative Joint Disease

doi: 10.1016/j.btre.2026.e00946

Figure Lengend Snippet: Functional neutralization of NGF binding to the TrkA receptor and SP-induced mast cell degranulation by antibodies elicited by rNGFSP in dogs. A) Inhibition of NGF–TrkA binding. Neutralization of NGF binding to the TrkA receptor was assessed by ELISA. Biotinylated NGF was incubated with TrkA in the presence of pooled sera from non-immunized dogs at day 0 (D0), sera from rNGFSP-immunized dogs at day 56 (D56), or a commercial monoclonal anti-NGF antibody (0.5 µg/mL). NGF alone served as a positive control. Sera were diluted 1:10 prior to the assay.. * p < 0.05. B) Inhibition of SP-induced mast cell degranulation. Flow cytometry analysis of mast cells stimulated with SP conjugated to BSA in the presence of IgG from non-immunized (D0) or immunized (D56) dogs. Representative plots show avidin–FITC-positive degranulating cells. The percentage of degranulating mast cells from replicate experiments is summarized on the right. All data are expressed as mean ± SEM, with * p < 0.05 compared to Day 0, determined by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Confocal microscopy of mast cell degranulation. Mast cells were stained with avidin–FITC (green) and DAPI (blue). SP or the positive control compound 48/80 (10 µM) induced granule release, whereas IgG from immunized dogs (D56) reduced SP-induced degranulation. Scale bar: 10 µm.

Article Snippet: After stimulation, cells were centrifuged (1000 × g, 10 min, 4 °C), resuspended in 150 μL PBS, and stained with 2 μg/mL Avidin-FITC (Thermo Scientific, #A821) for 20 min at room temperature.

Techniques: Functional Assay, Neutralization, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Flow Cytometry, Avidin-Biotin Assay, Confocal Microscopy, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Infiltration of CD68-positive activated microglia/macrophages co-expressing PSAP and PGRN into the SFO and surrounding tissues a – b) Double immunofluorescent staining of PGRN (green) and CD68 (red) around the SFO in 10-month-old-female WT and SAP-D −/− mice. b , Magnified images of the indicated white squares in a . ⅰ: SFO, ⅱ: Fornix, and ⅲ: Perivascular, bv: blood vessel. c ) Quantification of PGRN- and/or CD68-staining in the SFO and surrounding areas in WT and SAP-D −/− mice. Data are shown as the mean ± SD (n = 3). The left panel presents a stacked bar chart, whereas the right panel shows the individual data values in a bar chart format. d – h ) Triple immunofluorescent staining of PSAP (red), PGRN (green), and CD68 (cyan) around the SFO in 10-month-old-female SAP-D −/− mice. e) Magnified images of the indicated white squares in d) ⅳ: Boundary, ⅴ: Fornix, and ⅵ: Perivascular. White arrowheads indicate triple co-staining with PSAP, PGRN, and CD68. Open arrowheads indicate PGRN signals alone. Co-localization rates of CD68 positive areas in PSAP ( f ), PGRN ( g ), and PSAP-PGRN staining areas ( h ) around the SFO of SAP-D −/− mice, respectively. f-h ) The left panel presents a stacked bar chart, whereas the right panel presents the individual data values in a bar chart format. Data are shown as mean ± SD (n = 3). Nuclei are labeled by DAPI (blue) staining. All scale bars, 50 μm.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: c-Fos expression was induced in the SFO of SAP-D −/− mice without dehydration. a) Expression levels of c-Fos , Gpr37 , and Cd68 in the SFO and fornix were quantified via RT-qPCR. For RNA extraction, tissue samples were microdissected from the brains of 6-month-old female WT mice (n = 10) and SAP-D −/− mice (n = 6) under a stereomicroscope. After cDNA synthesis, RT-qPCR was performed, and the data were normalized to Gapdh expression. A significant increase in Cd68 expression was observed in SAP-D −/− mice, consistent with immunostaining results, confirming accurate sampling of the SFO and surrounding fornix. The results of Student's t-tests for each panel are as follows: left panel, p = 0.0046, Cohen's d = 1.74 (95 % CI: 0.0027, 0.0122); center panel, p = 0.0352, Cohen's d = 1.20 (95 % CI: 0.00091, 0.02191); and right panel, p = 0.0007, Cohen's d = 2.41 (95 % CI: 0.0079, 0.0233). b) Immunofluorescent staining of c-Fos (green) in the SFO of 10-month-old female WT and SAP-D −/− mice. WT and SAP-D −/− mice had free access to drinking water (indicated as FD) or 24 h water deprivation (indicated as DH). The white dotted lines enclose the SFO. Nuclei are labeled by DAPI (blue) staining. Scale bars, 50 μm. c) Percentage of c-Fos positive cells among all DAPI stained cells in the SFO (%). Data are shown as the mean ± SD (n = 7). , , , and indicate the individual values in each group. Two-way ANOVA showed significant main effects of water deprivation, F(1,24) = 35.13, p < 0.0001, ηp 2 = 0.13, 95 % CI (−8.67, −4.19), and genotype, F(1,24) = 130.40, p < 0.0001, ηp 2 = 0.36, 95 % CI (−14.64, −10.16), as well as a significant water deprivation × genotype interaction, F(1,24) = 38.60, p < 0.0001, ηp 2 = 0.14, 95 % CI (−17.98, −9.01). Post-hoc Tukey's tests showed significant differences between WT-FD versus WT-DH ( p < 0.0001, Cohen's d = 5.83 [95 % CI: −17.42, −8.94]), WT-FD versus SAP-D −/− -FD ( p < 0.0001, Cohen's d = 6.91 [95 % CI: −23.38, −14.91]), and WT-DH versus SAP-D −/− -DH ( p = 0.006, Cohen's d = 1.90 [95 % CI: −9.89, −1.41]), but no significant difference between SAP-D −/− -FD versus SAP-D −/− -DH ( p = 0.997, Cohen's d = 0.09 [95 % CI: −3.92, 4.54]). ns: no significant difference. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, RNA Extraction, cDNA Synthesis, Immunostaining, Sampling, Staining, Labeling