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: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells. Colors shown in the key and dotted lines denote gating used for analysis throughout this publication. b.) Scatterplot of DNA content versus median cyclin B1 intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. c.) Scatterplot of DNA content versus mean EdU intensity in MCF10A cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs. Colors shown in the key and dotted lines denote cyclin B1 gating used for analysis throughout this article. d.) Quantification of mean cyclin B1 intensity in MCF10A cells with non-targeting or cyclin B1 knockdowns to demonstrate cyclin B1 antibody specificity. A Mann-Whitney test was conducted to assess significance. Error bars are representative of 10-90 percentile range. P value: <0.0001. e.) Quantification of median cyclin B1 cytoplasmic intensity across the cell cycle from RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. f.) Quantification of integrated SLBP intensity in MCF10A cells with non-targeting or SLBP knockdowns to demonstrate SLBP antibody specificity. Error bars are representative of 10-90 percentile range. A Mann-Whitney test was conducted to assess significance. P value: <0.0001. g.) Breakdown of quantification of median SLBP nuclear intensity across the cell cycle from into individual graphs. Data are presented as mean ± SEM. h.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 5μM RO-3306, or a combination of 5μM AZD6738 and 5μM RO-3306 for 1hr (n=3 biological replicates). Data are presented as mean ± SEM. i.) Quantification of median SLBP nuclear intensity across the cell cycle from MCF10A cells treated with DMSO, 5μM AZD6738, 1μM NU6140, or a combination of 5μM AZD6738 and 1μM NU6140 for 1hrs (n=3 biological replicates). Data are presented as mean ± SEM.

Article Snippet: Human hTERT RPE-1 cells (ATCC, CRL-4000) were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, A5256801), 100U/mL penicillin/streptomycin, and 10μg/mL hygromycin B (Roche, 10843555001).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

doi: 10.64898/2026.05.07.723638

Figure Lengend Snippet: a.) Quantification of median cyclin B1 cytoplasmic intensity in RPE-1 cells with low or high yH2AX across the cell cycle. Cells were treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. b.) Quantification of mean yH2AX nuclear intensities across the cell cycle from control and cyclin B1 knockdown RPE-1 cells treated with DMSO, 5μM AZD6738, or 2μM LY2603618 for 16hrs (n=3 biological replicates). Data are presented as mean ± SEM. c.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM KU60019 + 5μM AZD6738, 5μM KU60019 + or 2μM LY2603618 + 5μM KU60019 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. d.) Quantification of mean yH2AX nuclear intensity across the cell cycle from MCF10A cells treated with DMSO + DMSO, DMSO + 5μM AZD6738, DMSO + 2μM LY2603618, 5μM KU60019 + DMSO, 5μM NU7441 + 5μM AZD6738, 5μM NU7441 + or 2μM LY2603618 + 5μM NU7441 for 7hrs (n=3 biological replicates). Data are presented as mean ± SEM. e.) Representative images of cells undergoing normal mitosis versus mitotic failure. Scale bar is equal to 15μm.

Article Snippet: Human hTERT RPE-1 cells (ATCC, CRL-4000) were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, A5256801), 100U/mL penicillin/streptomycin, and 10μg/mL hygromycin B (Roche, 10843555001).

Techniques: Control, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Age-dependent induction of ER stress in retinal pigment epithelium impairs phagocytosis via ADAM17-dependent MERTK shedding

doi: 10.1016/j.jbc.2026.111397

Figure Lengend Snippet: Pharmacological induction of ER stress attenuates phagocytic activity in cultured RPE cells. A , schematic diagram of the phagocytosis assay using fluorescein isothiocyanate (FITC)- and pHrodo succinimidyl ester (pHrodo)-conjugated photoreceptor outer segments (POS). B , a representative image of engulfed FITC-POS ( green ) and Hoechst 33,342 ( blue ) with plasma membrane staining 6 h after FITC-POS treatment. The plasma membrane ( gray ) was visualized by PlasMem Bright Red. Scale bar = 10 μm. C , a representative image of pHrodo signal ( yellow ), LAMP1 (magenta) at 24 h after pHrodo-POS treatment. Scale bar = 10 μm. D–F , Tunicamycin (Tm)-induced short-term ER stress reduces phagocytic activity in ARPE-19 and human primary RPE (hRPE) cells. D , experimental timeline for the assays shown in ( E ) and ( F ). E , quantification of fluorescence intensity for FITC-POS and pHrodo-POS in ARPE-19. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ### p < 0.001 vs. control (Cont) group (Dunnett’s test). F , quantitative data of fluorescence intensity for pHrodo-POS in hRPE cells. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ## p < 0.01, ### p < 0.001 vs. Cont group (Dunnett’s test). G , quantification of phagocytized pHrodo-POS after co-treatment with thapsigargin (Tg). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. # p < 0.05 vs. Cont group (Student's t test). H , cell death rate following Tm or Tg treatment for 6 h. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. N.S. > 0.05 vs. Cont group (Dunnett’s test). I – K , long-term ER stress reduces phagocytic activity in ARPE -19 and hRPE. I , experimental timelines for assays shown in ( J ) and ( K ). Quantitative data of fluorescence intensity of pHrodo-POS in ARPE-19 ( J ) and hRPE ( K ). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Cont group (Dunnett’s test).

Article Snippet: The human-derived RPE cell line, ARPE-19, was purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Cell Culture, Phagocytosis Assay, Clinical Proteomics, Membrane, Staining, Fluorescence, Control

Journal: The Journal of Biological Chemistry

Article Title: Age-dependent induction of ER stress in retinal pigment epithelium impairs phagocytosis via ADAM17-dependent MERTK shedding

doi: 10.1016/j.jbc.2026.111397

Figure Lengend Snippet: Maturation of ADAM17 mediated by Ca 2+ release from inositol - 1,4,5-trisphosphate receptors contributes to MERTK shedding and dysfunction of POS uptake. A , time-dependent change of the mature form of ADAM17 (matADAM17) in ARPE-19 cells after Tm treatment at 10 μg/ml. Data are presented as mean ± SEM (n = 4). Each point represents one independent sample prepared from separate wells. # p < 0.05 vs. Cont group (Welch's t test). B , expression level of matADAM17 in ARPE-19 after Tm treatment at 1 μg/ml for 54 h. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ### p < 0.001 vs. Cont group (Welch’s t test). C , localization of ADAM17 after Tm treatment at 10 μg/ml for 3 h. Representative images of ADAM17 ( yellow ), Golgin-97 ( magenta ), and Hoechst 33,342 ( blue ). Scale bar = 10 μm. Quantitative data of fluorescence intensity of ADAM17 colocalized with Golgin-97 after 1 and 3 h after Tm treatment. Data are presented as mean ± SEM (Cont; n = 97 cells, Tm 1 h; n = 103 cells, Tm 3 h; n = 104 cells). ### p < 0.001 vs. Cont group (Dunnett's T3 test). D – F , expression level of matADAM17 after Tm treatment at 10 μg/ml in the presence of decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) (100 μM, D), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) (100 μM, E ), or 2-aminoethoxydiphenyl borate (2-APB) (100 μM, F ). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ## p < 0.01, ### p < 0.001 vs. Cont group; ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Tm-only treated group (Games–Howell test). G , schematic diagram of ER stress-induced ADAM17 maturation and MERTK shedding. H and I , effect of ADAM17 small interfering RNA (siRNA) treatment on Tm-induced MERTK downregulation in ARPE-19. H , representative immunoblots of MERTK (extracellular domain), ADAM17, and β-actin. I , quantitative data for MERTK (extracellular domain). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ## p < 0.01 vs. control siRNA (siCont) single-treated group; ∗ p < 0.05 vs. siCont and Tm co-treated group (Student's t test). J and K , effect of ADAM17 siRNA on Tm-induced dysfunction of POS uptake in ARPE-19. J , schematic protocol of the POS uptake assay and ( K ) quantitative data of fluorescence intensity of FITC-POS internalized in RPE cells. Data are presented as mean ± SEM (n = 8). Each point represents one independent well. # p < 0.05 vs. siCont single-treated group; †† p < 0.01 vs. siCont and chloroquine co-treated group; ∗ p < 0.05 vs. siCont, chloroquine, and Tm co-treated group (Kruskal–Wallis test followed by post hoc Bonferroni test).

Article Snippet: The human-derived RPE cell line, ARPE-19, was purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Fluorescence, Small Interfering RNA, Western Blot, Control

Journal: Frontiers in Chemistry

Article Title: Bulky PP1 analogs exert cellular effects independently from analog-sensitive kinase inhibition

doi: 10.3389/fchem.2026.1812827

Figure Lengend Snippet: PP1 analogs inhibit cell migration. Cell migration of hTERT-RPE1 (left), HaCaT (middle) and MDA-MB-231 cells (right) was assessed by monitoring wound closure every 2 h over 48 h, in presence of various concentrations of PP1 inhibitors. Cytochalasin D, a potent actin polymerization inhibitor, was used as a reference inhibitor of cell migration . Relative wound cellular density was determined in 3 or 4 different wells. Histograms showing relative cellular densities after 12, 24, 48 h of treatments are shown in .

Article Snippet: We grew hTERT-RPE1 (telomerase-immortalized human retinal pigment epithelium cells; ATCC CRL-4000), HaCaT (immortalized human keratinocytes; AddexBio T0020001), and MDA-MB-231 (human breast adenocarcinoma cells; ATCC HTB-26) in DMEM medium (Gibco, 61965-059) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270-106) unless otherwise indicated.

Techniques: Migration