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(A) Human myotube senescent model. To induce the senescence of human <t>MSCs,</t> the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.
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(A) Human myotube senescent model. To induce the senescence of human <t>MSCs,</t> the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.
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(A) Human myotube senescent model. To induce the senescence of human <t>MSCs,</t> the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.
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(A) Human myotube senescent model. To induce the senescence of human <t>MSCs,</t> the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.
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(A) Human myotube senescent model. To induce the senescence of human <t>MSCs,</t> the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.
Supplementmix Msc Growth Medium 2, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(A) Human myotube senescent model. To induce the senescence of human <t>MSCs,</t> the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.
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(A) Human myotube senescent model. To induce the senescence of human MSCs, the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.

Journal: PLOS One

Article Title: An in vitro model to study molecular pathogenesis of sarcopenia established by a SASP-dependent human myotube culture

doi: 10.1371/journal.pone.0326968

Figure Lengend Snippet: (A) Human myotube senescent model. To induce the senescence of human MSCs, the cells were treated with doxorubicin for 24 hours and then removed. After 14 days, the conditioned medium of the d-Sen MSC culture was added to human myotubes differentiated from myoblasts (Hu5) for 7 days. (B) A representative Hoechst and γH2AX staining images of human MSCs treated with or without doxorubicin (scale bar, 250 μm). (C) Quantification of fluorescence intensity (γH2AX/Hoechst). (D) Gene expression of senescence-related markers by real-time PCR. Culture of human MSCs with or without doxorubicin. ** p < 0.01. (E) Gene expression of SASP markers by real-time PCR. Culture of human MSCs with or without doxorubicin. **p < 0.01. (F) Representative image of Hu5 cells differentiation. (scale bar, 500 μm). (G) Gene expression of muscle differentiation markers by real-time PCR. The days indicate as duration of culturing of Hu5 cells with DM. *p < 0.05, ** p < 0.01. Statistical analyses were conducted using the student’s t -test.

Article Snippet: Human Mesenchymal Stem Cells (MSCs) from Takara Bio were cultured with Mesenchymal Stem Cell Growth Medium 2 (Takara Bio) in early passages and then cultured to be acclimated in 4.5g/L high glucose Dulbecco modified Eagle medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; JRH Biosciences) and 1% penicillin/streptomycin (FUJIFILM Wako Pure Chemical Corporation).

Techniques: Staining, Fluorescence, Gene Expression, Real-time Polymerase Chain Reaction