Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: Variant positioning within the structural domains suggests differential impacts on WISP3 activity. ( A ) Structural architecture of WISP3 presented within the structure available in the AlphaFold database (AF- O95389 -F1-v4). These domains (IGFBP, TSP-1, and CTCK) tend to have higher confidence scores (higher pLDDT), indicating plausible structures for FoldX-based stability analyses. The IGFBP N-terminal domain has been isolated from the whole structure (44th to 117th residues based on Uniprot ID: O95389 ), and the positions of some critical amino acids are shown. ( B ) Schematic overview of WISP3 domain organization in wild-type and variant forms. Missense variants p.Gly83Glu and p.Cys114Trp are indicated by vertical dashed lines and asterisks within the IGFBP domain, reflecting single amino acid substitutions without truncation. In contrast, nonsense variants p.Cys52* and p.Tyr109* result in premature stop codons, leading to loss of downstream domains to different extents.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Variant Assay, Activity Assay, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: Verification of WISP3 variant expression in human chondrocytes. Human chondrocytes were transfected with plasmids encoding either wild-type WISP3 (Overexpression) or CCN6 variants (p.Cys52*, p.Tyr109*, p.Gly83Glu, and p.Cys114Trp). ( A ) Relative CCN6 mRNA expression levels were measured by qPCR and normalized to ACTB . Each condition was analyzed using four independent biological replicates, with two technical replicates per sample ( n = 8). In box plots, the central line represents the median; box limits correspond to the 25th and 75th percentiles. Whiskers indicate the range of data, excluding outliers, which are shown as individual points. ( B ) Protein expression was assessed by Western blot using an anti-HA antibody. The full-length WISP3 protein was detected at approximately 41 kDa. β-Tubulin was used as a loading control. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Significance is represented as ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Variant Assay, Expressing, Transfection, Over Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: WISP3 variant expression alters wound healing capacity and cell viability in human chondrocytes. ( A ) Representative phase-contrast images from wound healing assays captured at 0 h and 24 h following scratch induction, which was performed 48 h post-transfection. Images were acquired at 4× magnification; scale bars = 250 µm ( n = 3). ( B ) Quantification of open wound area at 24 h post-transfection, presented as a percentage of the initial wound area at 0 h. Box plots show the distribution of values; the horizontal line within each box represents the median, while the black diamonds indicate the mean. ( C ) Cell viability was evaluated 48 h post-transfection using the MTT assay. Absorbance was measured at 570 nm, and horizontal lines represent median values, which were used to compare metabolic activity across groups ( n = 16). Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Significance is represented as * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Variant Assay, Expressing, Transfection, MTT Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: Evaluation of chondrocyte adhesion and extracellular matrix (ECM) remodeling in response to WISP3 variants. Human chondrocytes were transfected with pcDNA3.1, wild-type, p.Cys52*, p.Tyr109*, p.Gly83Glu, p.Cys114Trp. ( A ) Representative images from the cell adhesion assay performed on human type I collagen-coated wells. Cells were fixed and stained with crystal violet after seeding (scale bar = 250 μm). ( B ) Spectrophotometric quantification of attached cells based on crystal violet absorbance, normalized to control ( n = 3). ( C ) Densitometric quantification of MMP-13 activity from zymography, presented as percentage activity normalized to the control condition ( n = 6). ( D ) Representative zymogram showing pro-MMP-13 band intensity across experimental groups. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Significance is represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Transfection, Cell Adhesion Assay, Staining, Control, Activity Assay, Zymography

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: Mitochondrial ROS and ER stress marker expression upon CCN6 variations. To investigate the impact of CCN6 variants on mitochondrial oxidative stress and ER stress responses in chondrocytes, flow cytometry and qPCR analyses were performed. ( A , B ) Flow cytometry histograms showing mitochondrial mass assessed by MitoTracker Green FM (left) and mitochondrial ROS (mtROS) levels determined using MitoSOX Red staining (right) with fluorescence intensity shifts reflecting changes in mean fluorescence intensity (MFI) across conditions ( n = 2). ( C ) Quantification of the mtROS/mass ratio, derived from MFI values and normalized to the pcDNA3.1 group (set as 1), illustrating differences in redox balance between variants. ( D ) Relative expression levels of ER stress markers CHOP and s/uXBP1 , determined by qPCR. Data are represented with error bars indicating SD ( n = 6). ( E ) CHOP/sXBP1 expression ratio calculated as an indicator of ER stress severity ( n = 6). Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Significance is represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Marker, Expressing, Flow Cytometry, Staining, Fluorescence, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: WISP3 localization and co-localization with mitochondria are differentially affected by disease-associated variants. ( A ) Representative confocal images showing subcellular localization of WISP3 (red), mitochondrial marker TOM20 (green), and nuclei (DAPI, blue) in chondrocytes transfected with either wild-type WISP3 or CCN6 variants. Merged images indicate the extent of co-localization. Scale bar: 10 μm. ( B ) Quantification of WISP3-TOM20 co-localization using Pearson correlation coefficient (R 2 ) reveals significantly reduced correlation in p.Cys52* compared to other groups. ( C ) Manders’ M2 coefficient, representing the fraction of WISP3 signal overlapping with TOM20. Each data point represents an individual cell (≥25 cells per condition from 3 biological replicates). Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Significance is represented as * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Marker, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: To examine the transcriptomic impact of disease-associated CCN6 variants, human chondrocytes were transfected with wild-type WISP3 (overexpression), p.Cys52*, or p.Cys114Trp constructs, and whole-transcriptome sequencing was performed. Transcriptomic profiling of CCN6 variants p.Cys52* and p.Cys114Trp in human chondrocytes. ( n = 2) ( A , B ) Volcano plots illustrating DEGs in p.Cys52* ( A ) and p.Cys114Trp ( B ) variant-overexpressing chondrocytes compared to wild-type WISP3-overexpressing cells. Red and blue dots represent significantly upregulated and downregulated genes, respectively (adjusted p < 0.05, |log 2 FC| > 1). ( C , D ) Gene ontology (GO) enrichment analysis of significantly regulated genes in p.Cys52* ( C ) and p.Cys114Trp ( D ). Color intensity reflects statistical significance (adjusted p -value), and dot size corresponds to the number of genes per GO term. ( E ) Heatmap showing significantly altered gene expressions across p.Cys52*, overexpression, and p.Cys114Trp groups, with hierarchical clustering. Functional grouping of genes related to unfolded protein response, mitochondrial metabolism, and cytoskeletal organization is indicated below. Z-score represents normalized gene expression.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Transfection, Over Expression, Construct, Sequencing, Variant Assay, Functional Assay, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Molecular Consequences of CCN6 Variants Encoding WISP3 in Progressive Pseudorheumatoid Dysplasia

doi: 10.3390/ijms26188838

Figure Lengend Snippet: Proteomic comparison of wild-type WISP3 and variants reveals distinct interaction landscapes. ( A , B ) Volcano plots showing differentially enriched proteins in chondrocytes expressing p.Tyr109* ( A ) and p.Cys114Trp ( B ) compared to wild-type WISP3 (OE). Blue and red indicate significantly downregulated and upregulated proteins, respectively. ( C ) Venn diagram illustrating the number of shared and unique interactors among overexpression of WISP3 (OE), p.Tyr109*, and p.Cys114Trp conditions. ( D ) Protein–protein interaction network constructed from LC–MS/MS–based co-immunoprecipitation data. Nodes represent proteins selectively or commonly pulled down with each construct. Color coding indicates functional categories (e.g., cytoskeletal, ER-associated, mitochondrial, ribosomal, UPR-related). Interactors are grouped by their presence in one or more conditions as indicated in the legend.

Article Snippet: The plasmid template containing the full-length human WISP3 cDNA ORF clone (Cat. No. HG15729-NY) was obtained from Sino Biological (Beijing, China).

Techniques: Comparison, Expressing, Over Expression, Construct, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Functional Assay