Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 4. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) RNA-seq analyses in human SMSCs after static and CTS treatment [(A1) volcano plot of differentially expressed genes (DEGs) in CTS group versus static group; (A2) KEGG pathway enrichment, red boxes represent Hippo and TGFβ signaling]. (B) Volcano plot of differentially expressed genes in regenerated tissue of PCL scaffold + synovium transplant group versus native porcine menisci [(B1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (B2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (B3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (C) Heatmap of differential gene cluster in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (C2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (C3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (D) KEGG pathway enrichment in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(D1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (D2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (D3) PCL scaffold + synovium transplant group at 2 versus 4 months, the red boxes represent Hippo and TGFβ signaling]. (E) Protein and mRNA levels in human SMSCs after static and CTS treatment [(E1) Western blot analysis; (E2) semiquantitative analysis of Western blot, n = 3, unpaired t test; (E3) mRNA levels, n = 8, unpaired t test]. (F) Cell immunofluorescence of human SMSCs after static and CTS treatment [(F1) cell immunofluorescence; (F2) six regions are randomly selected for semiquantitative analysis; the intensity of immunofluorescence is calculated by ImageJ software, n = 6, unpaired t test] (continued with Fig. 5). **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Software

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 5. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) Protein and mRNA levels in human SMSCs after being treated with dihy- drexidine and SIS3 [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (A3) the alcian blue staining; (A4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (A5) cell immunofluorescence; (A6) the semiquantitative analysis of cell immunofluorescence, Dihy represents dihydrexidine, n = 6, one-way ANOVA; (A7) the mRNA levels, n = 8, one-way ANOVA]. (B) Immunofluorescent colocalization of YAP and Smad2/3 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) immunofluorescent colocalization of YAP and Smad2/3; (B2) immu- nofluorescence overlap coefficient, n = 9]. (C) Immunofluorescent colocalization of pSmad2/3 and SOX9 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) immunofluorescent colocalization of pSmad2/3 and SOX9; (C2) immunofluorescence overlap coefficient, n = 9]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 6. Piezo1 mediates mechanotransduction on YAP-pSmad2/3-SOX9 axis through concerted activation of calcineurin and NFATc1 during MSCs chondro- genesis. (A) Protein and mRNA levels of Piezo1 in human SMSCs after static and CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A3) the cell immunofluorescence; (A4) the semiquantitative analysis of cell immunofluorescence, n = 6, unpaired t test; (A5) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of Piezo1 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (C) Protein and mRNA levels in human SMSCs after being treated with YODA1 or GsMTx-4 [(C1) the Western blot analysis; (C2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (C3) the mRNA levels, n = 8, unpaired t test; (C4) the alcian blue staining; (C5) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (C6) the cell immunofluorescence; (C7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. (D) Assessment of intracellular calcium content in human SMSCs [(D1) intracellular calcium content after static and CTS treatment; (D2) the semiquantitative analysis of calcium content, n = 5, unpaired t test; (D3) intracellular calcium content after being treated with YODA1 or GsMTx-4; (D4) the semiquantitative analysis of calcium content, n = 5, one-way ANOVA]. (E) Protein and mRNA levels in human SMSCs after CsA treatment [(E1) Western blot analysis, the red arrow represents dephosphorylated NFATc1, the blue arrows represent phosphorylated NFATc1; (E2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (E3) the alcian blue staining; (E4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (E5) the mRNA levels, n = 8, unpaired t test; (E6) the cell immunofluorescence; (E7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 7. The effect of matrix stiffness on YAP-pSmad2/3-SOX9 axis. (A) Protein and mRNA levels in human SMSCs after being treated with soft or stiff matrix [(A1) the mechanical properties of silicon rubber and polystyrene culture plate, n = 3; (A2) Western blot analysis, the red arrow indicates dephosphorylated NFATc1, the blue arrows indicate phosphorylated NFATc1; (A3) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A4) the alcian blue staining; (A5) the semiquantitative analysis of alcian blue staining, n = 3, unpaired t test; (A6) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of FAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) the immunofluorescence of FAK; (B2) the semiquantitative analysis of FAK, n = 6, one-way ANOVA]. (C) Immunofluorescent assessment of pFAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) the immunofluorescence of pFAK; (C2) the semiquantitative analysis of pFAK, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 8. The biomechanical stimulus and matrix stiffness regulate the expression of collagen cross-link enzymes (LOX and LH) through Piezo1. (A) Protein and mRNA levels of LOX and LH in human SMSCs after static or CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (A3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (A4) the cell immunofluorescence of LOX; (A5) the semiquantitative analysis of LOX immunofluorescence, n = 6, unpaired t test; (A6) the cell immunofluorescence of LH2; (A7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, unpaired t test; (A8) the mRNA levels, n = 8, unpaired t test]. (B) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with YODA1 or GsMTx-4 [(B1) the Western blot analysis; (B2) the semiquantitative analysis of LOX, n = 3, one-way ANOVA; (B3) the semiquantitative analysis of LH2, n = 3, one-way ANOVA; (B4) the cell immunofluorescence of LOX; (B5) the semiquantitative analysis of LOX immunofluorescence, n = 6, one-way ANOVA; (B6) the cell immunofluorescence of LH2; (B7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, one-way ANOVA; (B8) the mRNA levels, n = 8, one-way ANOVA]. (C) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with soft or stiff matrix [(C1) the Western blot analysis; (C2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (C3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (C4) the mRNA levels, n = 8, unpaired t test]. (D) Immunofluorescent assessment of LOX expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (E) Immunofluorescent assessment of LH2 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Comparison of human rRNA removal specificity and efficiency between the RTR2D procedure and NEBNext® rRNA Depletion kit. Human total RNA (1.0 µg) was subjected to the RTR2D (R2D) (with the pooled rRNA probes) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p < 0.001, compared with that of the NP group or the Input group. ( C ) Effect of rRNA removal protocols on the expression of housekeeping genes GAPDH and β-ACTIN ( a ) and genes/lncRNA with different abundances, c-MYC , TP53 and lncRNA HOTAIR ( b ). All qPCR reactions were done in triplicate.

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Comparison of mouse rRNA removal specificity and efficiency between the RTR2D procedure and NEB Next rRNA Depletion kit. Mouse total RNA (1.0 µg) was subjected to the RTR2D (R2D) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p < 0.001, compared with that of the NP group or the Input group. ( C ) Effect of rRNA removal protocols on the expression of housekeeping genes Gapdh and β-Actin ( a ) and genes/lncRNA with different abundances, c-Myc , Tp53 and lncRNA Hotair ( b ). All qPCR reactions were done in triplicate.

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Transcriptomic comparison of the RNA-seq libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols. Human total RNA (1.0 µg) was subjected to the rRNA removal process by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis (in replicate). ( A ) The average valid reads (RPKM) for the RTR2D ( a ) and the NEB kit ( b ) are depicted in various categories of transcripts. ( B ) Scatter plots and correlations of mRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a & b ). ( C ) Scatter plots and correlations of lncRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a & b ).

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: RNA Sequencing Assay

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Nutlin3A-induced transcriptomic changes determined by RNA-seq analysis of the NGS libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols . Exponentially growing human osteosarcoma line SJSA1 cells were treated with 2 µM Nutlin3A or DMSO control for 24 h and subjected to total RNA isolation. Human total RNA (1.0 µg) was subjected to rRNA removal by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis. ( A ) Scatter plots of mRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 54) and down-regulated transcripts ( d ) (total relative reads > 13). ( B ) Scatter plots of lncRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Significant up and down-regulated transcripts were defined as log2FoldChange > 1 and < −1, respectively, with a false detection rate (FDR) < 0.001. Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 540) and down-regulated transcripts ( d ) (total relative reads > 20).

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: RNA Sequencing Assay, Isolation, Generated