Journal: International journal of molecular sciences

Article Title: CTHRC1 Expression Results in Secretion-Mediated, SOX9-Dependent Suppression of Adipogenesis: Implications for the Regulatory Role of Newly Identified CTHRC1 + /PDGFR-Alpha + Stromal Cells of Adipose.

doi: 10.3390/ijms26051804

Figure Lengend Snippet: Figure 4. SOX9 Expression Is Indispensable to the Anti-Adipogenic Activity of hCTHRC1 Condi- tioned Medium In Vitro. (A–E) 3T3-L1 cells were seeded on Day-3 with either βgal or hCTHRC1 conditioned medium at a 1/4 dilution. Whole-cell lysates were collected on Day 0 to determine SOX9 expression by qPCR (A) and Western blot (B) analyses. (A) Average fold expression differences in Sox9 mRNA levels relative to housekeeping Gtf2b expression levels from five independent experiments (n = 5; ** p ≤0.01). Green symbols (triangle, circle, diamond, open square, and closed sqaure) are paired according to experimental replication. (C) Average SOX9 protein fold change densitometry values relative to housekeeping GTF2B protein expression levels from three independent experiments (n = 3; ** p ≤0.01). Green symbols (triangle, open circle, and closed circle) are paired according to experimental replication. (D,E) Representative confocal microscopy images of SOX9 protein lo- calization on Day 0 in 3T3-L1 cells treated with either βgal conditioned medium (D) or hCTHRC1 conditioned medium (E): nuclei (blue); SOX9 (green); F-actin/Alexa Fluor 546 Phalloidin (red). Scale bar: 20 µm. (F–H) 3T3-L1 cells were lentivirally transduced with either an shRNA construct targeting Sox9 mRNA (i.e., shSOX9), or a non-targeting scrambled control shRNA construct (i.e., shSCR). The resultant shSOX9 and shSCR cells were seeded on Day-3 with βgal or hCTHRC1 conditioned medium at a dilution of 1/60. Whole-cell lysates were collected from cohorts of shSOX9 and shSCR cells on Day 0 to assess SOX9 protein expression levels by Western blot analysis (G), while the other cohorts were chemically stimulated to undergo adipogenic differentiation for a total period of 6 days (F). (F) Representative Oil Red O quantification data. shSOX9 and shSCR cells were formalin fixed on

Article Snippet: Following blocking, primary antibodies against SOX9 (Cell Signaling Technology, Danvers, MA, USA; Cat. # 82630) were added at a 1/250 dilution in immunofluorescence blocking buffer and kept overnight at 4 °C.

Techniques: Expressing, Activity Assay, In Vitro, Western Blot, Confocal Microscopy, Transduction, shRNA, Construct, Control

Journal: International journal of molecular sciences

Article Title: CTHRC1 Expression Results in Secretion-Mediated, SOX9-Dependent Suppression of Adipogenesis: Implications for the Regulatory Role of Newly Identified CTHRC1 + /PDGFR-Alpha + Stromal Cells of Adipose.

doi: 10.3390/ijms26051804

Figure Lengend Snippet: Figure 5. Rho and Rac1 Signaling Mediate the SOX9-Dependent, Anti-Adipogenic Function of hCTHRC1 Conditioned Medium In Vitro. (A–E) 3T3-L1 cells were seeded on Day-3 with βgal or hCTHRC1 conditioned medium at a 1/4 dilution in the absence or presence of the combined application of NSC 23766 (N; 10 µM) and Y-27632 (Y; 15 µM) (N+Y). (A–D) Representative confocal microscopy images of SOX9 protein localization on Day 0 in 3T3-L1 cells treated with either βgal conditioned medium (A,C) or hCTHRC1 conditioned medium (B,D) in the absence (A,B) or presence (C,D) of the combined application of N (10 µM) and Y (15 µM): nuclei (blue); SOX9 (green); F- actin/Alexa Fluor 546 Phalloidin (red). Scale bar: 20 µm. (E) Quantification of SOX9+ nuclei based on 10 separate fields per experiment (n = 3; ** p ≤0.01). Not statistically significant (ns). (F–I) 3T3-L1 cells were seeded on Day-3 with βgal or hCTHRC1 conditioned medium at a dilution of 1/60, in the absence or presence of the combined application of N (3 µM) and Y (5 µM) (N+Y). Whole-cell lysates were collected from cohorts of cells on Day 0 to assess SOX9 protein expression levels by Western blot analysis (H), while the other cohorts were chemically stimulated to undergo adipogenic differentiation for a total period of 6 days (F). (F) Representative Oil Red O quantification data. 3T3-L1 cells were formalin fixed on Day 6 and stained with Oil Red O, which was then eluted and its concentration determined by absorbance spectroscopy. Per experiment, 3T3-L1 cells were plated in 24-well plates in which 6 wells each were treated with βgal or hCTHRC1 conditioned medium at a dilution of 1/60 in the presence or absence of N+Y (n = 4; ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001). (G) Graphical representation quantifying that hCTHRC1 conditioned medium has a significantly

Article Snippet: Following blocking, primary antibodies against SOX9 (Cell Signaling Technology, Danvers, MA, USA; Cat. # 82630) were added at a 1/250 dilution in immunofluorescence blocking buffer and kept overnight at 4 °C.

Techniques: In Vitro, Confocal Microscopy, Expressing, Western Blot, Staining, Concentration Assay, Spectroscopy

Journal: International journal of molecular sciences

Article Title: CTHRC1 Expression Results in Secretion-Mediated, SOX9-Dependent Suppression of Adipogenesis: Implications for the Regulatory Role of Newly Identified CTHRC1 + /PDGFR-Alpha + Stromal Cells of Adipose.

doi: 10.3390/ijms26051804

Figure Lengend Snippet: Figure 6. PDGFR-alpha+ Stromal Cells Express CTHRC1 In Vivo. (A) Cthrc1, Mfap5, and Pdgfra mRNA expression levels in inguinal white adipose tissue (iWAT) during postnatal development. Koza and colleagues [21] conducted microarray analyses of RNA isolated from the iWAT of C57BL/6 male mice aged 5, 10, 21, and 56 days, in which Cthrc1 and Mfap5 gene expression were shown to decrease in iWAT during early postnatal development. (B) qPCR analysis of Cthrc1 gene expression in iWAT isolated from 7-day-old and 3-month-old C57BL/6 male mice. Data were normalized to housekeeping Gtf2b expression levels and are presented as the average fold value per mouse (n = 4; ** p ≤0.01). Black diamonds refer to the relative Cthrc1 fold value per mouse. (C) qPCR analysis of Sox9 gene expression in iWAT isolated from 7-day-old wildtype and Cthrc1-null C57BL/6 littermate mice that were derived from heterozygous breeding pairs (three litters in total). Data were normalized to housekeeping Gtf2b expression levels and are presented as the average fold value per mouse (n = 5; * p ≤0.05). Black symbols (triangle, circle, diamond, open square, and closed square) are paired according to experimental replication comparing a littermate wildtype mouse to a Cthrc1-null mouse. (D–J) Representative multi-parameter flow cytometry workflow, displayed in the form of contour plots, assessing endogenous CTHRC1 protein expression in CD24+ versus PDGFR-alpha+ stromal

Article Snippet: Following blocking, primary antibodies against SOX9 (Cell Signaling Technology, Danvers, MA, USA; Cat. # 82630) were added at a 1/250 dilution in immunofluorescence blocking buffer and kept overnight at 4 °C.

Techniques: In Vivo, Expressing, Microarray, Isolation, Gene Expression, Derivative Assay, Flow Cytometry

Journal: International journal of molecular sciences

Article Title: CTHRC1 Expression Results in Secretion-Mediated, SOX9-Dependent Suppression of Adipogenesis: Implications for the Regulatory Role of Newly Identified CTHRC1 + /PDGFR-Alpha + Stromal Cells of Adipose.

doi: 10.3390/ijms26051804

Figure Lengend Snippet: Figure 7. Identification of CTHRC1 and SOX9 Gene Expression in PDGFRA+:MFAP5+ Enriched Cell Populations in Human Subcutaneous White Adipose Tissue. (A) Harmonized UMAP projection

Article Snippet: Following blocking, primary antibodies against SOX9 (Cell Signaling Technology, Danvers, MA, USA; Cat. # 82630) were added at a 1/250 dilution in immunofluorescence blocking buffer and kept overnight at 4 °C.

Techniques: Gene Expression

Journal: International journal of molecular sciences

Article Title: CTHRC1 Expression Results in Secretion-Mediated, SOX9-Dependent Suppression of Adipogenesis: Implications for the Regulatory Role of Newly Identified CTHRC1 + /PDGFR-Alpha + Stromal Cells of Adipose.

doi: 10.3390/ijms26051804

Figure Lengend Snippet: Figure 8. Conceptualizing the CTHRC1-SOX9 Signaling Axis Within White Adipose Tissue. Graphical illustration depicting white adipose tissue architecture and addressing the question of whether CTHRC1 is expressed in adipocyte progenitor cells and/or fibroblasts. Within white adipose tissue, adipocytes reside in a highly vascularized niche which contains adipocyte progenitor cells and fibroblasts that are each positioned between adipocytes and adjacent to vascular adventitia [22]. Merrick and colleagues previously identified the reticular interstitium which is an anatomically distinct,

Article Snippet: Following blocking, primary antibodies against SOX9 (Cell Signaling Technology, Danvers, MA, USA; Cat. # 82630) were added at a 1/250 dilution in immunofluorescence blocking buffer and kept overnight at 4 °C.

Techniques: