Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 1. WISP1 is endogenously upregulated in primary human DHLF. WISP1 transcript and secreted protein are expressed in NHLF and DHLF. DHLF exhibit significantly higher (A) WISP1 mRNA transcript and (B) secreted WISP1 protein in the condition media, detected by RT-qPCR and ELISA, respectively. Each data point represents one donor-derived primary cell line for both NHLF (n = 5 donors) and DHLF (n = 5 donors), with each experiment performed in triplicates and represented as mean ± SD. Statistical analysis was performed using paired t-test and significance denoted as * p < 0.5 or ** p < 0.01 versus healthy NHLF, as shown.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 2. TGFβ induces WISP1 expression in primary lung and dermal fibroblasts. Stimulation with TGFβ (1 ng/mL) for 24 h significantly increases COL1A1, COL3A1, IL6, ACTA2, FN1, and WISP1 mRNA as compared to control in (A) NHLF, (B) DHLF, and (C) NHDF for 3 respective donors (n = 3/each) analyzed by RT-qPCR. Notably, TGFβ decreased CCL2 in NHLF and DHLF, except in NHDF. Each experiment was independently performed in triplicates with three biological replicates (n = 3) for each donor and represented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis vs. vehicle control for respective donors. * denotes p < 0.5, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001 versus the unstimulated control, as shown.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 3. WISP1 promotes a striking increase in primary human dermal fibroblast cell proliferation. (A) Treatment with WISP1 (1000 nM) shows a striking increase in cell proliferation (p < 0.0001 via one- way ANOVA with Tukey’s post hoc analysis) detected by increased pRb positive cells as compared to control in NHDF. Representative 10× images were captured with the top image representing Hoechst nuclear stain, while the bottom panel represents pRb green fluorescence. PDGF-BB (100 nM) was used as an internal positive control to ensure assay reliability. The PDGF-BB dose response curve is shown in the Supplementary Materials. (B) % pRb positive cells were calculated by dividing pRb positive cells over total number of cells calculated by nuclear Hoechst staining. Three independent experiments (n = 3) were performed in triplicates and represented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis versus the vehicle-control condition and significance denoted as * p < 0.5, **** p < 0.0001.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Control, Staining, Fluorescence, Positive Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 4. WISP1 increases CCL2 and IL6 in primary fibroblasts. Stimulation with WISP1 (1000 nM) for 24 h significantly increases CCL2 and IL6 gene expression in (A) NHLF, (B) DHLF, and (C) NHDF as compared to control. TGFβ (1 ng/mL) for 24 h served as a positive control and significantly increases

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Gene Expression, Control, Positive Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 5. Recombinant WISP1 synergistically induces inflammatory markers along with TGFβ. (A) Primary human fibroblasts were stimulated with TGFβ (1 ng/mL) for 24 h for initiation, followed by WISP1 (1000 nM) for another 24 h as per the experimental layout. CCL2 and IL6 mRNA expression was significantly increased in (B) non-diseased lung and (D) dermal fibroblasts, but not in (C) IPF- diseased lung fibroblasts. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis vs. vehicle control for respective donors. * denotes p < 0.5, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001 versus the unstimulated control, for three biological replicates (n = 3) and represented as mean ± SD.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Recombinant, Expressing, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 6. WISP1 fibrotic gene signature in dermal fibroblasts. The volcano plot illustrates fibroblast genes upregulated (red) and downregulated (blue) upon stimulation with either (A) WISP1 alone or (B) in conjugation with TGFβ as compared to vehicle control (PBS) in NHDF (n = 3). The plots representing statistically significant gene with |FC| > 1.5 and adj. p-value < 0.1 are plotted with the x-axis representing log2 fold change (FC) versus the y-axis −log10 p-value. (C) The heatmap represents pathways significantly associated with the corresponding treatment condition as percent of genes influenced and the list of genes used in the pathway analysis is highlighted in Table S8. (D) Scatterplot showing log2 fold change for the differentially expressed genes when comparing WISP1 + TGFβ treatment with WISP1 (x-axis) versus TGFβ (y-axis). (E) Venn diagram representing unique and overlapping set of genes between the WISP1, TGFβ, and WISP1 + TGFβ treatment paradigms. A comprehensive lists of genes is provided in the Supplementary Materials.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Conjugation Assay, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 7. WISP1 fibrotic gene signature in lung fibroblasts. The volcano plot illustrates fibroblast genes upregulated (red) and downregulated (blue) upon stimulation with either (A) WISP1 alone or (B) in conjugation with TGFβ as compared to vehicle control (PBS) in NHLF (n = 3). The plots representing statistically significant gene with |FC| > 1.5 and adj p-value < 0.1 are plotted with the x-axis representing log2 fold change (FC) versus the y-axis −log10 p-value. (C) The heatmap represents pathways significantly associated with the corresponding treatment condition as percent of genes influenced and the list of genes used in the pathway analysis is highlighted in Table S8. (D) Scatterplot showing log2 fold change for the differentially expressed genes when comparing WISP1 + TGFβ treatment with WISP1 (x-axis) versus TGFβ (y-axis). (E) Venn diagram representing unique and overlapping set of genes between the WISP1, TGFβ, and WISP1 + TGFβ treatment paradigms. A comprehensive lists of genes is provided in the Supplementary Materials.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Conjugation Assay, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 8. WISP1 fibrotic gene signature in IPF-diseased fibroblast. The volcano plot illustrates fibroblast genes upregulated (red) and downregulated (blue) upon stimulation with either (A) WISP1 alone or (B) in conjugation with TGFβ as compared to vehicle control (PBS) in DHLF (n = 3). The plots representing statistically significant gene with |FC| > 1.5 and adj p-value < 0.1 are plotted with the x-axis representing log2 fold change (FC) versus the y-axis −log10 p-value. (C) The heatmap represents pathways significantly associated with the corresponding treatment condition as percent of genes influenced and the list of genes used in the pathway analysis is highlighted in Table S8. (D) Scatterplot showing log2 fold change for the differentially expressed genes when comparing WISP1 + TGFβ treatment with WISP1 (x-axis) versus TGFβ (y-axis). (E) Venn diagram represents unique and overlapping set of genes between the WISP1, TGFβ, and WISP1 + TGFβ treatment paradigms. A comprehensive list of genes is provided in the Supplementary Materials.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Conjugation Assay, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 9. Schematic summary of WISP1-TGFβ crosstalk in regulation of fibrosis progression.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: