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: Animal Models and Experimental Medicine

Article Title: Baihe Zhimu Decoction ameliorates anxious depression via PI3K / AKT / mTOR pathway‐mediated neuroprotection and inhibition of hippocampal neuronal apoptosis

doi: 10.1002/ame2.70295

Figure Lengend Snippet: BZD‐containing serum protects hippocampal neurons against apoptosis through activation of the PI3K/AKT/mTOR pathway. (A) Hoechst 33342 staining (×200), scale bar: 100 μm. (B) Quantification of apoptosis by Annexin V‐FITC/PI flow cytometry ( n = 3). (C) Representative Western blot bands and quantification of cleaved caspase‐3, Bax, and Bcl‐2 ( n = 4). (D) Representative western blot and quantification of PI3K, AKT, mTOR and their phosphorylated forms ( n = 4). Data are presented as mean ± SD. ## p < 0.01 vs . control group; ** p < 0.01 vs . model group; △ p < 0.05, △△ p < 0.01 vs . BZD group.

Article Snippet: Cells were labeled with 5 μL Annexin V‐FITC and 5 μL PI (E‐CK‐A211, Elabscience) for 15 min at room temperature in the dark.

Techniques: Activation Assay, Staining, Flow Cytometry, Western Blot, Control

Journal: Advanced Science

Article Title: Phosphorylated STYK1 Promotes Gemcitabine Resistance via Autophagy‐Mediated Cancer Stemness in Pancreatic Cancer

doi: 10.1002/advs.202521488

Figure Lengend Snippet: STYK1 promotes gemcitabine resistance in pancreatic cancer cells. (A, B) PANC‐1 cells were treated with different concentrations of gemcitabine for the indicated time periods, and STYK1 mRNA expression was assessed by RT‐qPCR (n = 6). **** p < 0.0001 according to Student's t ‐test. (C) Western blotting analysis of STYK1 expression levels in human pancreatic cancer cell lines PANC‐1 and AsPC‐1, as well as in the gemcitabine‐resistant cell lines PANC‐1/GEM and AsPC‐1/GEM. D) RT‐PCR analysis of STYK1 mRNA levels in PANC‐1, AsPC‐1, PANC‐1/GEM and AsPC‐1/GEM cells. **** p < 0.0001 according to Student's t ‐test. (E) Representative images of IHC staining for STYK1 in gemcitabine‐sensitive and resistant pancreatic cancer PDX tumors tissues. (F–I) Cell viability of PANC‐1/GEM and AsPC‐1/GEM cells with stable STYK1 knockdown, as well as PANC‐1 and AsPC‐1 cells stably overexpressing STYK1, following treatment with different concentrations of gemcitabine for 72 h, as determined by CCK‐8 assay. (J) PANC‐1/GEM cells with stable STYK1 knockdown were treated with gemcitabine (5 µM) for different durations as indicated, and OD values were assessed using the CCK‐8 assay. **** p < 0.0001 according to Student's t ‐test. (K–M) Apoptosis of PANC‐1/GEM and AsPC‐1/GEM cells with stable STYK1 knockdown following gemcitabine treatment (5 µM for PANC‐1/GEM and 40 µM for AsPC‐1/GEM, 24 h), assessed using an Annexin V‐AbFluor 488/PI double staining Apoptosis Detection Kit. Annexin V‐positive cells indicate early apoptosis, whereas PI‐positive cells indicate late apoptosis/necrosis (n = 3). **** p < 0.0001 according to Student's t ‐test. (N) Representative images of resected tumors from nude mice in different groups (n = 5). O) Tumor weights of resected tumors in different groups of mice (n = 5). **** p < 0.0001 according to Student's t ‐test. ** p < 0.01; **** p < 0.0001 according to Student's t ‐test. (P) Tumor volume change curves in different groups of mice (n = 5); Tumor volume was measured every 5 days after cell injection and during gemcitabine treatment (25 mg/kg). **** p < 0.0001 according to Student's t ‐test. (Q) Representative H&E staining and Ki67 immunohistochemical staining images of resected tumor tissues (n = 5). Scale bar: 50 µm. Data were expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 according to Student's t ‐test.

Article Snippet: Apoptosis was assessed using an Annexin V‐FITC Apoptosis Detection Kit (Elabscience, E‐CK‐A252).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Analysis, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Knockdown, Stable Transfection, CCK-8 Assay, Double Staining, Injection, Staining, Immunohistochemical staining

Journal: Advanced Science

Article Title: Age‐Related Lamin B1 Deficiency Activates SLC7A11‐Dependent Disulfidptosis to Impair Osteogenesis via Intercepting Mitochondrial Fission

doi: 10.1002/advs.78203

Figure Lengend Snippet: Age‐related LMNB1 deficiency in MSCs impairs the osteogenesis of SOP by triggering actin cytoskeleton collapse. (A) Representative micro‐CT images of femoral trabecular bone from LMNB1 fl/fl and LMNB1 △Prx1 mice, with 3D reconstruction, and quantitative analysis of trabecular and cortical bone parameters in the femurs (n = 5 mice per group). (B) Histological sections of tibias from LMNB1 fl/fl or LMNB1 △Prx1 mice stained with H&E, Masson's trichrome, and OSX immunohistochemistry and quantitative analysis of Masson‐positive area and osteoblast number per bone surface (OB.N/BS). (C) Schematic diagram of the calvarial defect model (top). Representative micro‐CT images showing calvarial defect healing in LMNB1 fl/fl and LMNB1 △Prx1 mice and quantitative analysis of the calvarial defect areas (n = 5 mice per group). (D) ARS and ALP staining of MSCs from LMNB1 fl/fl or LMNB1 △Prx1 mice during osteogenic differentiation and quantitative analysis of ARS intensity and ALP activity. (E) Western blot analysis of RUNX2, OSX and COL I expression in MSCs from LMNB1 fl/fl or LMNB1 △Prx1 mice during osteogenic differentiation. (F) Volcano plot of RNA‐seq data comparing MSCs at passage 12 and MSCs at passage 4 during osteogenic differentiation. (G) Volcano plot of RNA‐seq data comparing SOP‐MSCs and HD‐MSCs during osteogenic differentiation. (H) Western blot analysis of LMNB1 expression in MSCs extracted from mice aged 2, 4, 8, 12, and 18 months during osteogenic differentiation. (I) Western blot analysis of LMNB1 expression in MSCs at passages 3, 6, 9, 12 and 15 during osteogenic differentiation. (J) Western blot analysis of LMNB1 expression in HD‐MSCs and SOP‐MSCs during osteogenic differentiation. (K) Representative immunofluorescence images showing LMNB1 expression in MSCs of bone tissues from HD subjects or SOP patients. (L) Regional association plot of the LMNB1 locus for osteoporosis (p value = 9×10^‐8). (M) ARS and ALP staining of osteogenic differentiating HD‐MSCs+sh‐NC, HD‐MSCs +sh‐LMNB1, SOP‐MSCs+ov‐NC and SOP‐MSCs+ov‐LMNB1. (N) Western blot analysis of RUNX2, OSX, COL I and LMNB1 expression in HD‐MSCs+sh‐NC, HD‐MSCs+sh‐LMNB1, SOP‐MSCs+ov‐NC and SOP‐MSCs+ov‐LMNB1 during osteogenic differentiation. (O) GO enrichment analysis of DEGs from two RNA‐seq datasets (SOP‐MSCs vs HD‐MSCs, P12 MSCs vs P4 MSCs). (P) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of HD‐MSCs+sh‐NC, HD‐MSCs+sh‐LMNB1, SOP‐MSCs+ov‐NC and SOP‐MSCs+ov‐LMNB1. (Q) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of MSCs treated with sh‐NC, sh‐LMNB1 or sh‐LMNB1+ jasplakinolide (10 nM). (R) ARS and ALP staining of MSCs treated with sh‐NC, sh‐LMNB1 or sh‐LMNB1+ jasplakinolide during osteogenic differentiation and quantitative analysis of ARS intensity and ALP activity. (S) Western blot analysis of RUNX2, OSX and COL I expression in MSCs treated with sh‐NC, sh‐LMNB1 or sh‐LMNB1+ jasplakinolide during osteogenic differentiation. The data are presented as the means ± standard deviations (SD), n = 9 (unless stated), * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MSCs were prepared for imaging as follows: fluorescent labeling of MSCs was achieved by FITC phalloidin (Solarbio, CA1620) at 100 nM and MitoTracker Deepred (Beyotime, C1034) at 200 nM.

Techniques: Micro-CT, Analysis, Staining, Immunohistochemistry, Activity Assay, Western Blot, Expressing, RNA Sequencing, Immunofluorescence, High Content Screening

Journal: Advanced Science

Article Title: Age‐Related Lamin B1 Deficiency Activates SLC7A11‐Dependent Disulfidptosis to Impair Osteogenesis via Intercepting Mitochondrial Fission

doi: 10.1002/advs.78203

Figure Lengend Snippet: SLC7A11 as the pivotal molecule in LMNB1 deficiency‐mediated dysfunction of SOP‐MSCs, (A) Venn diagram identifying transcriptional leaked genes by the overlap of genes upregulated in P12 MSCs and SOP‐MSCs and related to LAD‐detached and SE regions in SOP‐MSCs, and SLC7A11 was identified as the only pivotal gene. (B) SLC7A11 signal traces of ATAC‐seq and CUT&Tag (LMNB1, H3K27ac, BRD4, POL II, H3K9me2 and H3K9me3) data. The shadow in purple shows the LAD region, and the shadows in green, orange and blue show newly formed SE in LAD‐detachment regions. (C) Representative immunofluorescence images showing SLC7A11 expression in bone tissues from HD subjects or SOP patients. (D) Western blot analysis of LMNB1 and SLC7A11 expression in HD‐MSCs+sh‐NC, HD‐MSCs+sh‐LMNB1, SOP‐MSCs+ov‐NC and SOP‐MSCs+ov‐LMNB1 during osteogenic differentiation. (E) Representative immunofluorescence images showing LMNB1 and SLC7A11 expression in the tibia from LMNB1 fl/fl or LMNB1 △Prx1 mice. (F) H3K27ac and LMNB1 CUT&Tag‐qPCR analysis of LMNB1 fl/fl MSCs , LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+enCRISPRi‐1 and LMNB1 △Prx1 MSCs+enCRISPRi‐2 at enhancer‐1 and enhancer‐2. (G) Western blot analysis of LMNB1 and SLC7A11 expression in LMNB1 fl/fl MSCs , LMNB1 fl/fl MSCs+enCRISPRi‐1 and LMNB1 fl/fl MSCs+enCRISPRi‐2, LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+enCRISPRi‐1 and LMNB1 △Prx1 MSCs+enCRISPRi‐2 during osteogenic differentiation. (H) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+enCRISPRi‐1 and LMNB1 △Prx1 MSCs+enCRISPRi‐2 and actin morphology quantitative analysis of actin SER Ridge (n = 402–509 cells) and anisotropy (n = 50 cells). (I) ARS and ALP staining of LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+ enCRISPRi‐1, LMNB1 △Prx1 MSCs+ enCRISPRi‐2, and quantitative analysis of ARS intensity and ALP activity. (J) Western blot analysis of RUNX2, OSX and COL I expression in LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+enCRISPRi‐1 and LMNB1 △Prx1 MSCs+enCRISPRi‐2 during osteogenic differentiation. (K) Representative micro‐CT images of femoral trabecular bone from 18‐month SLC7A11 fl/fl and SLC7A11 △Prx1 mice, with 3D reconstruction, and quantitative analysis of trabecular and cortical bone parameters in the femurs (n = 5). (L) Histological sections of tibias from 18‐month SLC7A11 fl/fl and SLC7A11 △Prx1 mice stained with H&E, Masson's trichrome, and OSX immunohistochemistry and quantitative analysis of Masson‐positive area and OB.N/BS (n = 5 mice per group). (M) Representative micro‐CT images showing calvarial defect healing in 18‐month SLC7A11 fl/fl and SLC7A11 △Prx1 mice and quantitative analysis of the calvarial defect areas (n = 5 mice per group). (N) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of SLC7A11 fl/fl MSCs, SLC7A11 △Prx1 MSCs or SLC7A11 △Prx1 MSCs+CytoD (500 nM) and actin morphology quantitative analysis of actin SER Ridge (n = 336–613 cells) and anisotropy (n = 50 cells). (O) ARS and ALP staining of SLC7A11 fl/fl MSCs, SLC7A11 △Prx1 MSCs or SLC7A11 △Prx1 MSCs+ CytoD during osteogenic differentiation and quantitative analysis of ARS intensity and ALP activity. (P) Western blot analysis of RUNX2, OSX and COL I expression in SLC7A11 fl/fl MSCs, SLC7A11 △Prx1 MSCs or SLC7A11 △Prx1 MSCs+CytoD during osteogenic differentiation. The data are presented as the means ± standard deviations (SD), n = 9 (unless specifically stated), * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MSCs were prepared for imaging as follows: fluorescent labeling of MSCs was achieved by FITC phalloidin (Solarbio, CA1620) at 100 nM and MitoTracker Deepred (Beyotime, C1034) at 200 nM.

Techniques: Immunofluorescence, Expressing, Western Blot, Analysis, High Content Screening, Morphology, Staining, Activity Assay, Micro-CT, Immunohistochemistry

Journal: Advanced Science

Article Title: Age‐Related Lamin B1 Deficiency Activates SLC7A11‐Dependent Disulfidptosis to Impair Osteogenesis via Intercepting Mitochondrial Fission

doi: 10.1002/advs.78203

Figure Lengend Snippet: Disulfidptosis activation contributed to actin cytoskeleton collapse and impaired osteogenesis of SOP‐MSCs. (A) Cystine uptake, NADP + /NADPH ratios and non‐reducing and reducing western blot analysis of actin in SOP‐MSCs treated with 100 µM, 200 µM, 400 µM or 800 µM cystine. (B) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of SOP‐MSCs treated with 100 µM, 200 µM, 400 µM or 800 µM cystine and actin morphology quantitative analysis of the actin SER Ridge (n = 479–706 cells) and anisotropy (n = 50 cells). (C) ARS and ALP staining of SOP‐MSCs treated with 100 µM, 200 µM, 400 µM or 800 µM cystine and quantitative analysis of ARS intensity and ALP activity. (D) Western blot analysis of RUNX2, OSX and COL I expression in SOP‐MSCs treated with 100 µM, 200 µM, 400 µM or 800 µM cystine during osteogenic differentiation. (E) Cystine uptake, NADP + /NADPH ratios and non‐reducing and reducing western blot analysis of actin in HD‐MSCs and SOP‐MSCs. (F) Cystine uptake, NADP + /NADPH ratios and non‐reducing and reducing western blot analysis of actin in LMNB1 fl/fl MSCs and LMNB1 △Prx1 MSCs. (G) Cystine uptake, NADP + /NADPH ratios and non‐reducing and reducing western blot analysis of Actin in SLC7A11 fl/fl MSCs and SLC7A11 △Prx1 MSCs. (H) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of HD‐MSCs, SOP‐MSCs and SOP‐MSCs+NAC (2 mM) and actin morphology quantitative analysis of actin SER Ridge (n = 362–497 cells) and anisotropy (n = 50 cells). (I) ARS and ALP staining of HD‐MSCs, SOP‐MSCs and SOP‐MSCs+NAC and quantitative analysis of ARS intensity and ALP activity. (J) Western blot analysis of RUNX2, OSX, COL I and SLC7A11 expression in HD‐MSCs, SOP‐MSCs and SOP‐MSCs+NAC during osteogenic differentiation and relative quantitative analysis of RUNX2, OSX, COL I and SLC7A11 expression. The data are presented as the means ± standard deviations (SD), n = 9 (unless specifically stated), * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MSCs were prepared for imaging as follows: fluorescent labeling of MSCs was achieved by FITC phalloidin (Solarbio, CA1620) at 100 nM and MitoTracker Deepred (Beyotime, C1034) at 200 nM.

Techniques: Activation Assay, Western Blot, Analysis, Immunofluorescence, High Content Screening, Morphology, Staining, Activity Assay, Expressing

Journal: Advanced Science

Article Title: Age‐Related Lamin B1 Deficiency Activates SLC7A11‐Dependent Disulfidptosis to Impair Osteogenesis via Intercepting Mitochondrial Fission

doi: 10.1002/advs.78203

Figure Lengend Snippet: Conditional knockout of SLC7A11 in MSCs reverses disulfidptosis and restores bone mass in LMNB1 △Prx1 mice. (A) Schematic diagram of the dCKO construction procedure using AAV9‐Flex‐shSLC7A11. (B) Representative immunofluorescence images showing LMNB1 and SLC7A11 expression in tibias from LMNB1 △Prx1 or dCKO mice. (C) Cystine uptake, NADP + /NADPH ratios and non‐reducing and reducing western blot analysis of actin in LMNB1 fl/fl , LMNB1 △Prx1 or dCKO MSCs cultured with 200 µM cystine and LMNB1 △Prx1 or dCKO MSCs cultured with 800 µM cystine. (D) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of LMNB1 fl/fl , LMNB1 △Prx1 or dCKO MSCs cultured with 200 µM cystine and LMNB1 △Prx1 or dCKO MSCs cultured with 800 µM cystine, and actin morphology quantitative analysis of actin SER Ridge (n = 282–466 cells) and anisotropy (n = 50 cells). (E) ARS and ALP staining of LMNB1 fl/fl , LMNB1 △Prx1 or dCKO MSCs cultured with 200 µM cystine, LMNB1 △Prx1 or dCKO MSCs cultured with 800 µM cystine, and quantitative analysis of ARS intensity and ALP activity. (F) Western blot analysis of RUNX2, OSX, COL I, SLC7A11 and LMNB1 expression in LMNB1 fl/fl , LMNB1 △Prx1 or dCKO MSCs cultured with 200 µM cystine and LMNB1 △Prx1 or dCKO MSCs cultured with 800 µM cystine during osteogenic differentiation. (G) Representative micro‐CT images of femoral trabecular bone from LMNB1 △Prx1 and dCKO mice, with 3D reconstruction and quantitative analysis of trabecular and cortical bone parameters in the femurs (n = 5 mice per group). (H) Histological sections of tibias from LMNB1 △Prx1 and dCKO mice stained with H&E, Masson's trichrome, and OSX immunohistochemistry and quantitative analysis of Masson‐positive area and OB.N/BS (n = 5 mice per group). (I) Representative micro‐CT images showing calvarial defect healing in LMNB1 △Prx1 and dCKO mice and quantitative analysis of the calvarial defect areas (n = 5 mice per group). The data are presented as the means ± standard deviations (SD), n = 9 (unless specifically stated), * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MSCs were prepared for imaging as follows: fluorescent labeling of MSCs was achieved by FITC phalloidin (Solarbio, CA1620) at 100 nM and MitoTracker Deepred (Beyotime, C1034) at 200 nM.

Techniques: Gene Knockout, Immunofluorescence, Expressing, Western Blot, Analysis, Cell Culture, High Content Screening, Morphology, Staining, Activity Assay, Micro-CT, Immunohistochemistry

Journal: Advanced Science

Article Title: Age‐Related Lamin B1 Deficiency Activates SLC7A11‐Dependent Disulfidptosis to Impair Osteogenesis via Intercepting Mitochondrial Fission

doi: 10.1002/advs.78203

Figure Lengend Snippet: Disulfidptosis‐induced disulfide bond formation in actin disrupts DRP1‐mediated mitochondrial fission. (A) High‐content fluorescence imaging of MitoTracker Green and mitochondrial network analysis of LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP (1 mM). and classification of mitochondrial morphology and summed branch lengths mean of mitochondria. (B) Representative images of mitochondrial transmission electron microscopy in LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (C) Representative images of mt‐Keima in LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (D) Representative images of CellRox in LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (E) TMRE flow cytometry analysis of LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP and quantitative analysis of TMRE MFI. (F) ATP measurement of LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (G) OCR quantitative analysis of LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP (n = 3). (H) Western blot analysis showing DRP1 expression in the mitochondria of LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (I) Co‐IP analysis showing the binding of DRP1 and actin in LMNB1 fl/fl MSCs, LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (J) Representative images of MitoTracker DeepRed, FITC phalloidin and DRP1 immunofluorescence showing the colocalization of mitochondria, actin and DRP1 in LMNB1 fl/fl MSCs, LMNB1 △Prx1 MSCs or LMNB1 △Prx1 MSCs+TCEP. (K) ARS and ALP staining of LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+TCEP or LMNB1 △Prx1 MSCs+TCEP+Mdivi‐1 (10 µM) and quantitative analysis of ARS intensity and ALP activity. (L) Western blot analysis of RUNX2, OSX and COL I expression in LMNB1 △Prx1 MSCs, LMNB1 △Prx1 MSCs+TCEP or LMNB1 △Prx1 MSCs+TCEP+Mdivi‐1. The data are presented as the means ± standard deviations (SD), n = 9 (unless specifically stated), * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MSCs were prepared for imaging as follows: fluorescent labeling of MSCs was achieved by FITC phalloidin (Solarbio, CA1620) at 100 nM and MitoTracker Deepred (Beyotime, C1034) at 200 nM.

Techniques: Fluorescence, Imaging, Analysis, Morphology, Transmission Assay, Electron Microscopy, Flow Cytometry, Western Blot, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Immunofluorescence, Staining, Activity Assay

Journal: Advanced Science

Article Title: Age‐Related Lamin B1 Deficiency Activates SLC7A11‐Dependent Disulfidptosis to Impair Osteogenesis via Intercepting Mitochondrial Fission

doi: 10.1002/advs.78203

Figure Lengend Snippet: Naldemedine restores osteogenesis in SOP by targeting SLC7A11‐dependent disulfidptosis. (A) Molecular dynamics simulations of SLC7A11 and NAL. (B) Binding energy of the SLC7A11‐NAL complexes over time. (C) RMSD values of the SLC7A11‐NAL complexes over time. (D) Cystine uptake, NADP + /NADPH ratios and non‐reducing and reducing western blot analysis of actin in SOP‐MSCs+DMSO and SOP‐MSCs+NAL. (E) Representative TRITC‐Phalloidin immunofluorescence images captured by a high‐content analysis system showing the actin network texture of SOP‐MSCs+DMSO and SOP‐MSCs+NAL and actin morphology quantitative analysis of actin SER Ridge (n = 423–485 cells) and anisotropy (n = 50 cells). (F) High‐content fluorescence imaging of MitoTracker Green and mitochondrial network analysis of SOP‐MSCs+DMSO and SOP‐MSCs+NAL and classification of mitochondrial morphology and summed branch length mean of mitochondria. (G) Representative images of mitochondria transmission electron microscopy in SOP‐MSCs+DMSO and SOP‐MSCs+NAL. (H) Representative images of mt‐Keima in SOP‐MSCs+DMSO and SOP‐MSCs+NAL. (I) Representative images of CellRox in SOP‐MSCs+DMSO and SOP‐MSCs+NAL. (J) TMRE flow cytometry analysis of SOP‐MSCs+DMSO and SOP‐MSCs+NAL and quantitative analysis of TMRE MFI. (K) ATP measurement of SOP‐MSCs+DMSO and SOP‐MSCs+NAL. (L) OCR quantitative analysis of SOP‐MSCs+DMSO and SOP‐MSCs+NAL (n = 3). (M) ARS and ALP staining of SOP‐MSCs+DMSO and SOP‐MSCs+NAL and quantitative analysis of ARS intensity and ALP activity. (N) Western blot analysis of RUNX2, OSX and COL I expression in SOP‐MSCs+DMSO and SOP‐MSCs+NAL and relative quantitative analysis of RUNX2, OSX and COL I expression. (O) Representative micro‐CT images of femoral trabecular bone from SOP mice or SOP mice treated with NAL, with 3D reconstruction, and quantitative analysis of trabecular and cortical bone parameters in the femurs (n = 5 mice per group). (P) Histological sections of tibias from SOP mice or SOP mice treated with NAL stained with H&E, Masson's trichrome, and OSX immunohistochemistry and quantitative analysis of Masson‐positive area and OB.N/BS (n = 5 mice per group). (Q) Representative micro‐CT images showing calvarial defect healing in SOP mice or SOP mice treated with NAL and quantitative analysis of the calvarial defect areas (n = 5 mice per group). The data are presented as the means ± standard deviations (SD), n = 9 (unless specifically stated), * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: MSCs were prepared for imaging as follows: fluorescent labeling of MSCs was achieved by FITC phalloidin (Solarbio, CA1620) at 100 nM and MitoTracker Deepred (Beyotime, C1034) at 200 nM.

Techniques: Binding Assay, Western Blot, Analysis, Immunofluorescence, High Content Screening, Morphology, Fluorescence, Imaging, Transmission Assay, Electron Microscopy, Flow Cytometry, Staining, Activity Assay, Expressing, Micro-CT, Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Hydroxygenkwanin Identified in Rehmanniae Radix Praeparata–Corni Fructus Ameliorates Postmenopausal Osteoporosis via Modulation of the AGE-RAGE Signaling Pathway

doi: 10.2147/JIR.S608114

Figure Lengend Snippet: RAGE-related signaling is involved in the anti-osteoclastogenic effect of HGK. ( A ) Representative immunofluorescence images of p53 expression. ( B ) Representative immunofluorescence images of NFATc1, RAGE, and CTSK expression. ( C ) Western blot analysis of CTSK and NFATc1, with or without FPS-ZM1 treatment; blots are representative of three independent biological replicates (n = 3). ( D ) Representative images of F-actin ring formation visualized by phalloidin staining. Yellow dashed boxes indicate the regions shown at higher magnification in the corresponding adjacent panels; white arrows indicate representative positive staining signals. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the RANKL groups.

Article Snippet: Upon completion of osteoclast induction, cells were fixed and subjected to staining with FITC conjugated phalloidin (Solarbio, China) to visualize F-actin rings.

Techniques: Immunofluorescence, Expressing, Western Blot, Analysis, Staining