Journal: Journal of visualized experiments : JoVE

Article Title: CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

doi: 10.3791/59432

Figure Lengend Snippet: Materials

Article Snippet: lenti-CRISPRv2-blast , Addgene , 83480 , .

Techniques: Modification, Recombinant, Staining, Lysis, Gel Extraction, Plasmid Preparation, Injection, Transfection, SYBR Green Assay, Titration, Ligation

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 1 CRISPR/Cas9-mediated gene targeting of DGCR8 in hESCs. a Schematic diagram of DGCR8 targeting using CRISPR/Cas9-mediated genome editing with homologous recombination. Neo stands for neomycin-resistance gene cassette used for positive selection. Red triangle indicates the FRT site. b Western blotting analysis of DGCR8 with protein extracts from WT and DR8dex2 hESCs. β-actin was used as the loading control. c G-banded karyotyping analysis of WT and DR8dex2 hESCs. d Immunofluorescence analysis of pluripotency markers OCT4, SOX2, and NANOG in WT and DR8dex2 hESCs. Scale bar, 12.5 μm. e Immunofluorescence analysis of representative markers of three germ layers in teratomas derived from WT or DR8dex2 hESCs. Scale bar, 50 μm. f Clonal expansion analysis of WT and DR8dex2 hESCs. Data were presented as mean ± SEM. n = 3 wells per cell type, ns means no significance. g Cell cycle analysis of WT and DR8dex2 hESCs. n = 3 wells per cell type, ns means no significance. Statistical significances were assessed by a two-tailed unpaired Student’s t test

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: CRISPR, Homologous Recombination, Selection, Western Blot, Control, Derivative Assay, Cell Cycle Assay, Two Tailed Test

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 2 DGCR8dex2 accelerates cellular senescence in hMSCs. a FACS analysis of the MSC markers CD73, CD90, and CD105 in WT and DR8dex2 hMSCs. b Western blotting analysis of DGCR8 in WT and DR8dex2 hMSCs. β-actin was used as the loading control. c Growth curve of WT and DR8dex2 hMSCs. d Clonal expansion analysis of WT and DR8dex2 hMSCs at early passage (P3) and late passage (P6). Scale bar, 30 μm. Data were presented as mean ± SEM. n = 3 wells per cell type, ***p < 0.001. e SA-β-gal staining of WT and DR8dex2 hMSCs at early (P3) and late (P6) passages. Scale bar, 20 μm. Data were presented as mean ± SEM. n = 4 images per cell type, ***p < 0.001. f Western blotting analysis of P16, P21, and GATA4 in WT and DR8dex2 hMSCs. β-actin was used as the loading control. g Heatmap showing quantitative RT-PCR analysis of the indicated genes in P3- and P6-hMSCs. Expression levels of the indicated genes in DR8dex2 hMSCs were normalized to those in WT hMSCs. (h) Whole genome analysis of copy number variations (CNVs) in WT and DR8dex2 hMSCs. i Photon flux from tibialis anterior (TA) muscles of nude mice transplanted with WT (left) or DR8dex2 hMSCs (right) that express luciferase. The attrition of hMSCs after implantation was measured by the reduction of luciferase activity in TA muscles using an in vivo imaging system (IVIS). Data were presented as mean ± SEM. n = 3 mice per group, *p < 0.05. j SA-β-gal staining of DR8dex2 hMSCs transduced with lentiviruses expressing luciferase (Luc) or DGCR8. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. k Clonal expansion analysis of DR8dex2 hMSCs transduced with lentiviruses expressing Luc or DGCR8. Data were presented as mean ± SEM. n = 3 wells per condition, ***p < 0.001. Statistical significances were assessed by a two-tailed unpaired Student’s t test

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: Western Blot, Control, Staining, Quantitative RT-PCR, Expressing, Muscles, Luciferase, Activity Assay, In Vivo Imaging, Transduction, Two Tailed Test

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 3 DGCR8 regulates hMSC senescence in a miRNA processing-independent way. a Clonal expansion analysis of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8-mtDRBD. Scale bar, 20 μm. Data were presented as mean ± SEM. n = 3 wells per condition, ***p < 0.001. b Ki67 staining of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8-mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, *p < 0.05. c RT-qPCR detection of P16, IL6, and IL8 in DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8- mtDRBD. Data were presented as mean ± SEM. n = 3 wells per condition, **p < 0.01. d SA-β-gal staining of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8-mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. Scale bar, 20 μm. e Immunostaining of γH2AX and 53BP1 in DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8-mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, **p < 0.01. Scale bar, 10 μm. Box plots show the median (center line inside the box), upper and lower quartiles (bounds of box), largest and smallest values (whiskers)

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: Transduction, Expressing, Staining, Quantitative RT-PCR, Immunostaining

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 5 DGCR8 and its interacting proteins are required for the homeostatic maintenance of hMSCs. a Left, 3D construction of a z stack of H3K9me3 and Lamin B immunofluorescence images shown in supplementary Fig. 7i of WT and DR8dex2 hMSCs. Scale bar, 10 μm. Right, mean fluorescence intensity of H3K9me3 in supplementary Fig. 7i was measured by ImageJ. Data were presented as mean ± SEM. n = 60 cells per cell type, ***p < 0. 001. b SA-β-gal staining of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT (1-773), or DR8-Δ58. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. c Clonal expansion analysis of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, DR8-WT (1-773), or DR8- Δ58. Data were presented as mean ± SEM. n = 3 wells per condition, ***p < 0.001. d SA-β-gal staining of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, KAP1, Lamin B1, or HP1γ. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. e Clonal expansion analysis of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, KAP1, Lamin B1, or HP1γ. Data were presented as mean ± SEM. n = 3 wells per condition, **p < 0.01. f Ki67 staining of DR8dex2 hMSCs transduced with lentiviruses expressing Luc, KAP1, Lamin B1, or HP1γ. Data were presented as mean ± SEM. n = 4 images per condition, *p < 0.05. g Immunostaining of LAP2 and H3K9me3 in DR8dex2 hMSCs transduced with lentiviruses expressing Luc, KAP1, Lamin B1, or HP1γ. Scale bar, 10 μm. Mean fluorescence intensity of LAP2 was calculated. Data were presented as mean ± SEM. n = 120 cells per condition. ***p < 0.001. h Photon flux from tibialis anterior (TA) muscles of nude mice transplanted with DR8dex2 hMSCs with co-infection of lentiviral vectors expressing GFP or KAP1 (left)/Lamin B1 (middle)/HP1γ (right) and lentiviral vector expressing luciferase. Luciferase activity in the TA muscles was detected by an in vivo imaging system (IVIS) 4 days after implantation, demonstrating slower attrition of DR8dex2 hMSCs transduced with KAP1, Lamin B1, or HP1γ. Data were presented as mean ± SEM. n = 3 mice per group, *p < 0.05. Statistical significances were assessed by a two-tailed unpaired Student’s t test

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: Staining, Transduction, Expressing, Immunostaining, Muscles, Infection, Plasmid Preparation, Luciferase, Activity Assay, In Vivo Imaging, Two Tailed Test

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 6 DGCR8 is downregulated in senescent hMSCs and DGCR8 overexpression attenuates hMSC aging. a Heatmap showing the quantification of protein levels in young and old human primary MSCs. Expression levels of indicated proteins in old hMSCs were normalized to those in young hMSCs. b Western blotting analysis of DGCR8 in replicative senescent (RS) and WS hMSCs. Y, young, S, senescent. β-actin was used as the loading control. c SA-β-gal staining of RS hMSCs transduced with Luc, DR8-WT, or DR8-mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. Scale bar, 20 μm. d SA-β-gal staining of WS hMSCs transduced with Luc, DR8-WT, or DR8-mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. Scale bar, 20 μm. e SA-β-gal staining of primary hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8- mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, ***p < 0.001. f Ki67 staining of primary hMSCs transduced with lentiviruses expressing Luc, DR8-WT, or DR8-mtDRBD. Data were presented as mean ± SEM. n = 4 images per condition, **p < 0.01. Statistical significances were assessed by a two-tailed unpaired Student’s t test

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: Over Expression, Expressing, Western Blot, Control, Staining, Transduction, Two Tailed Test

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 8 Aging and inflammation factors dysregulated in mouse osteoarthritis are rescued by DGCR8 overexpression. a Quantitative RT-PCR analysis of P16, P21, Il6 and Mmp13 in non-invasive mouse (n = 3 mice) joints and ACLT joints treated with lentiviruses expressing Luc (n = 3 mice), DR8-WT (n = 3 mice) or DR8-mtDRBD (n = 5 mice). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. b Left, heatmap showing Z-score normalized expression levels of downregulated genes in non-invasive mouse joints and ACLT joints treated with lentiviruses expressing DR8-WT or DR8-mtDRBD, compared with ACLT joints treated with lentiviruses expressing Luc. Right, heatmap showing Z-score normalized expression levels of upregulated genes in non-invasive mouse joints and ACLT joints treated with lentiviruses expressing DR8-WT or DR8-mtDRBD, compared with ACLT joints treated with lentiviruses expressing Luc. The downregulated and upregulated genes were listed in Supplementary Data 7. Statistical significances were assessed by a two-tailed unpaired Student’s t test

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis.

doi: 10.1038/s41467-019-10831-8

Figure Lengend Snippet: Fig. 9 DGCR8 alleviates osteoarthritis of aged mice. a Schematic of the time course for experiments in b–d. b Bone density analysis of mouse joints. Data were presented as mean ± SEM. n = 5 mice per group, *p < 0.05. Box plots show the median (center line inside the box), upper and lower quartiles (bounds of box), largest and smallest values (whiskers). c Heatmap showing quantitative RT-PCR analysis of the indicated genes in aged mouse joints with lentivirus treatment. Expression levels of the indicated genes in aged mouse joints were normalized to Luc group. d Left, Immunohistochemistry of P16 expression in aged mouse joints with lentivirus treatment. Right, Statistical analysis of P16-positive cells. Scale bar, 100 μm. Data were presented as mean ± SEM. n = 5 mice per group, **p < 0.01. Statistical significances were assessed by a two-tailed unpaired Student’s t test

Article Snippet: Briefly, the sgRNA targeting DGCR8 or LMNB1 was cloned into lenti-CRISPRv2 (#52961, Addgene) with an hSpCas9 expression cassette (pLenti-CRISPRv2DGCR8 or pLenti-CRISPRv2-LMNB1).

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry, Two Tailed Test