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anti h3ps10 antibody  (Cytoskeleton Inc)


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    Cytoskeleton Inc anti h3ps10 antibody
    Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an <t>anti-H3pS10</t> antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .
    Anti H3ps10 Antibody, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 97/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/phalloidin/Acti-stain+555+phalloidin/pmc13657169-474-12-16
    Average 97 stars, based on 290 article reviews
    anti h3ps10 antibody - by Bioz Stars, 2026-10
    97/100 stars

    Images

    1) Product Images from "Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells"

    Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

    Journal: iScience

    doi: 10.1016/j.isci.2026.117658

    Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .
    Figure Legend Snippet: Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

    Techniques Used: Blocking Assay, Staining, Analysis

    Pex19 KO mESCs have reduced exit from pluripotency during the early stage of neural differentiation (A) Morphology of WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation. Scale barss, 100 μm. (B) Deletion of Pex19 delays the reduction of pluripotency genes during early neural differentiation. The expression of indicated genes was analyzed before and after three days of in vitro neural differentiation by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s., not significant). (C) The protein level of Nanog in WT and Pex19 KO cells after three days of in vitro neural differentiation. Scale bars, 20 μm. (D) Adding back PEX19 restores the differentiation-induced reduction of pluripotency protein in Pex19 KO cells. The levels of indicated proteins were analyzed in WT, Pex19 KO (KO), and Pex19 KO + PEX19 (KO + PEX19) 46C mESCs before and after three days of in vitro neural differentiation by immunoblotting. (E) Treatment with DMSO partially increases G1 phase in Pex19 KO cells after three days of in vitro neural differentiation. WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (F) Adding back PEX19 or treatment with DMSO partially rescues the differentiation defects in Pex19 KO cells. The levels of indicated genes were analyzed in WT, Pex19 KO (KO), or Pex19 KO + PEX19 (KO + P19) 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO by qPCR ( n = 3 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, and ∗∗∗∗q < 0.0001). (G) Treatment with DMSO partially restores the pluripotency exit defect in Pex19 KO cells. (H) Treatment with a low dose of nocodazole decreases the G1 population in WT cells after three days of in vitro neural differentiation. WT 46C mESCs before (D0) or after three days of in vitro neural differentiation with or without 12.5 ng/mL nocodazole were analyzed for G1 population as in (E) ( n = 3 biological replicates/group; data are presented as mean ± SEM; one-way ANOVA; ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (I) Treatment with a low dose of nocodazole delays the reduction of pluripotency genes in WT cells after three days of in vitro neural differentiation. The levels of indicated pluripotency genes were analyzed in WT 46C mESCs treated as in (H) ( n = 3 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗ q < 0.05, ∗∗ q < 0.01, ∗∗∗ q < 0.001, ∗∗∗∗ q < 0.0001, n.s., not significant). See also .
    Figure Legend Snippet: Pex19 KO mESCs have reduced exit from pluripotency during the early stage of neural differentiation (A) Morphology of WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation. Scale barss, 100 μm. (B) Deletion of Pex19 delays the reduction of pluripotency genes during early neural differentiation. The expression of indicated genes was analyzed before and after three days of in vitro neural differentiation by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s., not significant). (C) The protein level of Nanog in WT and Pex19 KO cells after three days of in vitro neural differentiation. Scale bars, 20 μm. (D) Adding back PEX19 restores the differentiation-induced reduction of pluripotency protein in Pex19 KO cells. The levels of indicated proteins were analyzed in WT, Pex19 KO (KO), and Pex19 KO + PEX19 (KO + PEX19) 46C mESCs before and after three days of in vitro neural differentiation by immunoblotting. (E) Treatment with DMSO partially increases G1 phase in Pex19 KO cells after three days of in vitro neural differentiation. WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (F) Adding back PEX19 or treatment with DMSO partially rescues the differentiation defects in Pex19 KO cells. The levels of indicated genes were analyzed in WT, Pex19 KO (KO), or Pex19 KO + PEX19 (KO + P19) 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO by qPCR ( n = 3 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, and ∗∗∗∗q < 0.0001). (G) Treatment with DMSO partially restores the pluripotency exit defect in Pex19 KO cells. (H) Treatment with a low dose of nocodazole decreases the G1 population in WT cells after three days of in vitro neural differentiation. WT 46C mESCs before (D0) or after three days of in vitro neural differentiation with or without 12.5 ng/mL nocodazole were analyzed for G1 population as in (E) ( n = 3 biological replicates/group; data are presented as mean ± SEM; one-way ANOVA; ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (I) Treatment with a low dose of nocodazole delays the reduction of pluripotency genes in WT cells after three days of in vitro neural differentiation. The levels of indicated pluripotency genes were analyzed in WT 46C mESCs treated as in (H) ( n = 3 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗ q < 0.05, ∗∗ q < 0.01, ∗∗∗ q < 0.001, ∗∗∗∗ q < 0.0001, n.s., not significant). See also .

    Techniques Used: Morphology, In Vitro, Expressing, Western Blot, Staining, Analysis

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    Article Title: Cu‐Enhanced Medium‐Entropy TiZrCoCrMo Alloys: Balancing Superior Antibacterial and Osteogenic Properties for Orthopedic Implants
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    Image Search Results


    Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

    Journal: iScience

    Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

    doi: 10.1016/j.isci.2026.117658

    Figure Lengend Snippet: Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

    Article Snippet: After fixation with 4% paraformaldehyde, cells were processed for immunofluorescence assay with anti-H3pS10 antibody and phalloidin (Cytoskeleton, PHDH1) staining.

    Techniques: Blocking Assay, Staining, Analysis

    Pex19 KO mESCs have reduced exit from pluripotency during the early stage of neural differentiation (A) Morphology of WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation. Scale barss, 100 μm. (B) Deletion of Pex19 delays the reduction of pluripotency genes during early neural differentiation. The expression of indicated genes was analyzed before and after three days of in vitro neural differentiation by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s., not significant). (C) The protein level of Nanog in WT and Pex19 KO cells after three days of in vitro neural differentiation. Scale bars, 20 μm. (D) Adding back PEX19 restores the differentiation-induced reduction of pluripotency protein in Pex19 KO cells. The levels of indicated proteins were analyzed in WT, Pex19 KO (KO), and Pex19 KO + PEX19 (KO + PEX19) 46C mESCs before and after three days of in vitro neural differentiation by immunoblotting. (E) Treatment with DMSO partially increases G1 phase in Pex19 KO cells after three days of in vitro neural differentiation. WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (F) Adding back PEX19 or treatment with DMSO partially rescues the differentiation defects in Pex19 KO cells. The levels of indicated genes were analyzed in WT, Pex19 KO (KO), or Pex19 KO + PEX19 (KO + P19) 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO by qPCR ( n = 3 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, and ∗∗∗∗q < 0.0001). (G) Treatment with DMSO partially restores the pluripotency exit defect in Pex19 KO cells. (H) Treatment with a low dose of nocodazole decreases the G1 population in WT cells after three days of in vitro neural differentiation. WT 46C mESCs before (D0) or after three days of in vitro neural differentiation with or without 12.5 ng/mL nocodazole were analyzed for G1 population as in (E) ( n = 3 biological replicates/group; data are presented as mean ± SEM; one-way ANOVA; ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (I) Treatment with a low dose of nocodazole delays the reduction of pluripotency genes in WT cells after three days of in vitro neural differentiation. The levels of indicated pluripotency genes were analyzed in WT 46C mESCs treated as in (H) ( n = 3 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗ q < 0.05, ∗∗ q < 0.01, ∗∗∗ q < 0.001, ∗∗∗∗ q < 0.0001, n.s., not significant). See also .

    Journal: iScience

    Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

    doi: 10.1016/j.isci.2026.117658

    Figure Lengend Snippet: Pex19 KO mESCs have reduced exit from pluripotency during the early stage of neural differentiation (A) Morphology of WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation. Scale barss, 100 μm. (B) Deletion of Pex19 delays the reduction of pluripotency genes during early neural differentiation. The expression of indicated genes was analyzed before and after three days of in vitro neural differentiation by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s., not significant). (C) The protein level of Nanog in WT and Pex19 KO cells after three days of in vitro neural differentiation. Scale bars, 20 μm. (D) Adding back PEX19 restores the differentiation-induced reduction of pluripotency protein in Pex19 KO cells. The levels of indicated proteins were analyzed in WT, Pex19 KO (KO), and Pex19 KO + PEX19 (KO + PEX19) 46C mESCs before and after three days of in vitro neural differentiation by immunoblotting. (E) Treatment with DMSO partially increases G1 phase in Pex19 KO cells after three days of in vitro neural differentiation. WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (F) Adding back PEX19 or treatment with DMSO partially rescues the differentiation defects in Pex19 KO cells. The levels of indicated genes were analyzed in WT, Pex19 KO (KO), or Pex19 KO + PEX19 (KO + P19) 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO by qPCR ( n = 3 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, and ∗∗∗∗q < 0.0001). (G) Treatment with DMSO partially restores the pluripotency exit defect in Pex19 KO cells. (H) Treatment with a low dose of nocodazole decreases the G1 population in WT cells after three days of in vitro neural differentiation. WT 46C mESCs before (D0) or after three days of in vitro neural differentiation with or without 12.5 ng/mL nocodazole were analyzed for G1 population as in (E) ( n = 3 biological replicates/group; data are presented as mean ± SEM; one-way ANOVA; ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (I) Treatment with a low dose of nocodazole delays the reduction of pluripotency genes in WT cells after three days of in vitro neural differentiation. The levels of indicated pluripotency genes were analyzed in WT 46C mESCs treated as in (H) ( n = 3 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗ q < 0.05, ∗∗ q < 0.01, ∗∗∗ q < 0.001, ∗∗∗∗ q < 0.0001, n.s., not significant). See also .

    Article Snippet: After fixation with 4% paraformaldehyde, cells were processed for immunofluorescence assay with anti-H3pS10 antibody and phalloidin (Cytoskeleton, PHDH1) staining.

    Techniques: Morphology, In Vitro, Expressing, Western Blot, Staining, Analysis

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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