Journal: Journal of Orthopaedic Translation

Article Title: Targeting FGFR3 signaling and drug repurposing for the treatment of SLC26A2-related chondrodysplasia in mouse model

doi: 10.1016/j.jot.2023.09.003

Figure Lengend Snippet: Inhibition of Fgfr3 in chondrocytes ameliorates chondrocyte proliferation apoptosis and differentiation impairment in mouse growth plate chondrocytes due to Slc26a2 deficiency (A) (B) Ki67 immunostaining and TUNEL staining (red) of tibial sections of P0 mice. Scale bar: 50 ​μm (C) (D) Immunofluorescent analyses of FGFR3 signaling downstream mediators p-ERK1/2 and p-STAT1 (red) in proliferating chondrocytes of tibia sections of P0 mice. Scale bar: 50 ​μm (E) Immunostaining of Col X (red) of tibial sections of P0 mice (F) Quantification of Ki67-labeled cells, TUNEL-labeled cells, and p-ERK1/2- and p-STAT1-positive cells in the growth plate (n ​= ​9/group). For all the above-mentioned statistical analyses, significance was determined by One-way ANOVA followed by Tukey's multiple comparisons test, and the results are shown as the mean ​± ​SD, ∗ P ＜0.05, ∗∗ P ＜0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following primary antibodies used were Ki67 (Millipore, Burlington, MA, USA), anti-Col X (Abclonal, A11645, 1:100), anti-phosphatase-ERK1/2 (Cell Signaling Technology, 4370, 1:100), anti-phosphatase-STAT1 (Cell Signaling Technology, 9172, 1:100), anti-SOX9 (Abcam, ab185966, 1:100), anti-Col2α1 (Thermo Fisher, PA1-26206, 1:100), and anti-aggrecan (Thermo Fisher, MA3-16,888, 1:100).

Techniques: Inhibition, Immunostaining, TUNEL Assay, Staining, Labeling

Journal: Journal of Orthopaedic Translation

Article Title: Targeting FGFR3 signaling and drug repurposing for the treatment of SLC26A2-related chondrodysplasia in mouse model

doi: 10.1016/j.jot.2023.09.003

Figure Lengend Snippet: NVP-BGJ398 pharmacological intervention inhibits overactivation of the FGFR3 signaling pathway in Slc26a2 cKO mice (A) Safranin O and fast green staining of sections of tibial growth plates at P49. Slightly increased proliferating and hypertrophic chondrocytes were observed in NVP-BGJ398-treated growth plates compared with those of tamoxifen-induced chondrodysplasia groups. Scale bar: 100 ​μm (B) (C) Ki67 immunostaining and TUNEL staining (red) of tibia sections on P49. Scale bar: 50 ​μm (D) (E) Immunofluorescent analyses of FGFR3 signaling downstream mediators p-ERK1/2 and p-STAT1 (red) in growth plate chondrocytes on tibia sections at P49. Scale bar: 50 ​μm (F) Immunostaining of Col X (red) on tibia sections (G) Quantification of Ki67-labeled cells, TUNEL-labeled cells, and p-ERK1/2- and p-STAT1-positive cells in the growth plate (n ​= ​7). For all the above-mentioned statistical analyses, significance was determined by One-way ANOVA followed by Tukey's multiple comparisons test, and the results are shown as the mean ​± ​SD, ∗P＜0.05, ∗∗P＜0.01, ns: not statistically significant.

Article Snippet: The following primary antibodies used were Ki67 (Millipore, Burlington, MA, USA), anti-Col X (Abclonal, A11645, 1:100), anti-phosphatase-ERK1/2 (Cell Signaling Technology, 4370, 1:100), anti-phosphatase-STAT1 (Cell Signaling Technology, 9172, 1:100), anti-SOX9 (Abcam, ab185966, 1:100), anti-Col2α1 (Thermo Fisher, PA1-26206, 1:100), and anti-aggrecan (Thermo Fisher, MA3-16,888, 1:100).

Techniques: Staining, Immunostaining, TUNEL Assay, Labeling

Journal: eLife

Article Title: Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

doi: 10.7554/eLife.96941

Figure Lengend Snippet: ( A ) Experimental scheme depicting the generation of CRISPR/Cas9-edited mesenchymal sword of Damocles bone morphogenetic type-2 (MSOD-B) lines, and the subsequent in vitro and in vivo tissue formation assessment. ( B ) Overview of the native human VEGF coding sequence composed of eight exons. Designed guide RNAs (gRNAs) and their targeted binding sites are illustrated, as well as the corresponding expected impact on the coding sequence. gRNAs targeting exon 1 (orange) disrupt translation initiation and inhibit protein expression, gRNA targeting exon 2 (blue) disrupts VEGF receptor binding, and gRNA targeting exon 8 (green) alters the C-terminal sequence and represses activation of protein. ( C ) ELISA-based quantitative analysis of VEGF protein content in cell culture supernatant from expanded single cell colonies. From all clones, only two had no detectable level of VEGF (1.3_3 and 1.3_6). These clones were subsequently defined as MSOD-BΔV1 and MSOD-BΔV2. ( D ) Histological assessment of living and lyophilized in vitro differentiated constructs using Safranin O staining (scale bars=100 µm and 20 µm for magnified areas). Both the MSOD-B and MSOD-BΔV1 displayed glycosaminoglycans (GAG) (orange to reddish in Safranin O), indicating successful cartilage formation. Left bottom inserts show the whole tissue section. ( E ) Quantitative assessment of the total GAG content in MSOD-B and MSOD-BΔV1 in vitro differentiated constructs, post-lyophilization. Unpaired t-test, n=10 biological replicates, ***p<0.001. ( F ) ELISA-based quantitative assessment of VEGF protein in in vitro differentiated constructs, post-lyophilization. Unpaired t-test, n=3–4 biological replicates, ****p<0.0001. ( G ) Immunofluorescence images of MSOD-B and MSOD-BΔV1 tissues, post-lyophilization. Displayed images consist of 3D-stacks from 80- to 100-µm-thick sections, stained for VEGF (yellow), Collagen Type X (COLX, red), and Collagen Type I (COL1, gray). A clear reduction in the VEGF signal could be observed in the MSOD-BΔV1 tissues, indicating a successful VEGF knockdown (scale bars=80 µm). All error bars in the figures indicate the standard deviation (SD). Panel A was created with BioRender.com .

Article Snippet: After blocking/permeabilization, sections were stained with primary antibodies: mouse anti-Collagen II (Invitrogen, MA137493), Anti-Collagen 1 (Abcam, ab138492), VEGF polyclonal antibody (Bioss Antibodies, bs-0279R), rabbit anti-Collagen Type X (ColX) (abbexa, abx101469), Goat anti mouse/rat CD31 (R&D Systems, Cat# 11-0319-42), VEGF at 4°C overnight.

Techniques: CRISPR, In Vitro, In Vivo, Sequencing, Binding Assay, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Clone Assay, Construct, Staining, Lyophilization, Immunofluorescence, Knockdown, Standard Deviation

Journal: eLife

Article Title: Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

doi: 10.7554/eLife.96941

Figure Lengend Snippet: ( A ) Overview of the human RUNX2 coding sequence comprising eight exons. Guide RNAs (gRNAs) and their corresponding expected protein structure. The gRNA targeting exon 2 (orange) disrupts the DNA binding domain, gRNA targeting exon 5 (violet) disrupts nuclear translocation, gRNAs targeting exon 6 (blue) disrupt the transcriptional activation domain, and gRNAs targeting exon 8 (green) disrupt the nuclear matrix targeting signal and repress the protein function. ( B ) Intracellular flow cytometry for RUNX2 detection in mesenchymal sword of Damocles bone morphogenetic type-2 (MSOD-B) and RUNX2-edited clones. A clear protein reduction could be observed in the 6.1_1 and 6.1_23 clones. ( C ) Western blot analysis of RUNX2 in cultured MSOD-B and RUNX2-edited cells. The genetic editing of RUNX2 is confirmed by the detection of the truncated proteins. Actin is used as a control to normalize the protein content. ( D ) Histological analysis of living and lyophilized in vitro differentiated constructs using Safranin O staining (scale bars=100 µm and 20 µm for magnified areas), indicating the presence of cartilage matrices. ( E ) Quantitative assessment of the total GAG content in corresponding in vitro generated lyophilized tissues. Unpaired t-test, n=10–11 biological replicates **p<0.01. ( F ) Immunofluorescence images of MSOD-B and MSOD-BΔR1 lyophilized tissues. Displayed images consist of 3D-stacks from 80 to 100 µm thick sections, stained for DAPI (blue), Collagen Type II (COL2, yellow), and Collagen Type X (COLX, red). A clear reduction in the COLX signal could be observed in the MSOD-BΔR1 tissues, indicating impaired hypertrophy (scale bars=500 µm). ( G ) Isosurface-based quantification of the COL2 immunofluorescent signal using the IMARIS software. No significant difference between groups confirms the retention of cartilage formation in RUNX2-edited constructs. Unpaired t-test, n=3 biological replicates, n.s.=not significant. ( H ) Isosurface-based quantification of the COLX immunofluorescent signal using the IMARIS software, confirming the disruption of hypertrophy in the MSOD-BΔR1 constructs. Unpaired t-test, n=3, ***p<0.001. ( I ) Alizarin Red staining evidencing a lack of mineralization in the MSOD-BΔR1 culture compared to the MSOD-B. ( J ) Quantitative polymerase chain reaction (PCR) analysis displaying the relative expression levels of osteogenesis-related genes: RUNX2, COL1, and ALPL. The expression is normalized to GAPDH as housekeeping gene. n.d.=not detected. All error bars in the figures indicate either the standard deviation (SD) or the standard error of the mean (SEM); the specific metric used is consistent within each figure. Figure 3—source data 1. annotated western blot analysis of Runt-related transcription factor 2 (RUNX2) editing. Western blot analysis of RUNX2 in cultured mesenchymal sword of Damocles bone morphogenetic type-2 (MSOD-B) (MB) and RUNX2-edited cells (clones 6.1 and 6.23, respectively). The genetic editing of RUNX2 is confirmed by the detection of the truncated proteins. Actin is used as a control to normalize the protein content. Exposure was set at 13.7 s. Figure 3—source data 2. annotated western blot analysis of Runt-related transcription factor 2 (RUNX2) editing. Non-annotated western blot pictures of RUNX2 (left part of the gel) in cultured mesenchymal sword of Damocles bone morphogenetic type-2 (MSOD-B) (line 3) and RUNX2-edited cells (clone 6.1 line 4 and clone 6.23 line 5, respectively). The genetic editing of RUNX2 is confirmed by the detection of the truncated proteins. Actin (right part of the gel) is used as a control to normalize the protein content.

Article Snippet: After blocking/permeabilization, sections were stained with primary antibodies: mouse anti-Collagen II (Invitrogen, MA137493), Anti-Collagen 1 (Abcam, ab138492), VEGF polyclonal antibody (Bioss Antibodies, bs-0279R), rabbit anti-Collagen Type X (ColX) (abbexa, abx101469), Goat anti mouse/rat CD31 (R&D Systems, Cat# 11-0319-42), VEGF at 4°C overnight.

Techniques: Sequencing, Binding Assay, Translocation Assay, Activation Assay, Flow Cytometry, Clone Assay, Western Blot, Cell Culture, Control, In Vitro, Construct, Staining, Generated, Immunofluorescence, Software, Disruption, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: eLife

Article Title: Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

doi: 10.7554/eLife.96941

Figure Lengend Snippet: Representative immunofluorescence staining images of COL2, COLX, and DAPI tissue section of in vitro cartilage constructs show reduction of COLX expression in knockout construct (mesenchymal sword of Damocles [MSOD]-BΔR2), indicating reduction of hypertrophy (scale bars=500 µm).

Article Snippet: After blocking/permeabilization, sections were stained with primary antibodies: mouse anti-Collagen II (Invitrogen, MA137493), Anti-Collagen 1 (Abcam, ab138492), VEGF polyclonal antibody (Bioss Antibodies, bs-0279R), rabbit anti-Collagen Type X (ColX) (abbexa, abx101469), Goat anti mouse/rat CD31 (R&D Systems, Cat# 11-0319-42), VEGF at 4°C overnight.

Techniques: Immunofluorescence, Staining, In Vitro, Construct, Expressing, Knock-Out

Journal: eLife

Article Title: Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

doi: 10.7554/eLife.96941

Figure Lengend Snippet:

Article Snippet: After blocking/permeabilization, sections were stained with primary antibodies: mouse anti-Collagen II (Invitrogen, MA137493), Anti-Collagen 1 (Abcam, ab138492), VEGF polyclonal antibody (Bioss Antibodies, bs-0279R), rabbit anti-Collagen Type X (ColX) (abbexa, abx101469), Goat anti mouse/rat CD31 (R&D Systems, Cat# 11-0319-42), VEGF at 4°C overnight.

Techniques: CRISPR, Knock-Out, Transfection, Construct, Plasmid Preparation, Flow Cytometry, Western Blot, Control, Immunofluorescence, Immunostaining, Marker, Sequencing