Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Mouse subcutaneous adipose stromal cells (primary ASCs) differentiate into lipid-laden adipocytes after 10 days in culture. Images are oil red O (ORO) stained cells at day 0 (D0) or day 10 (D10) of adipocyte differentiation. Graph shows quantification of ORO accumulation over time. (B-D) Expression of adipocyte progenitor markers Cd34 (b), Pdgfra (c), and Cd24a (d) in mouse primary ASCs during proliferation (pro) or through differentiation. (E-F) Expression of preadipocyte markers Pparg (e) and Fabp4 (f) in mouse primary ASCs during proliferation (pro) or through differentiation. (G-H) Expression of Esr1 gene (g) or ERα protein (h) in mouse immortalized APCs during proliferation (pro) or through differentiation. (I) undifferentiated mouse primary ASCs treated with EtOH (Veh), E 2 , 4-OH tamoxifen (TAM) or E 2 +TAM. Images represent cells at day 4 of treatment. Scale=200µm. Inset graph represents cell number over the 4-day proliferation assay. Two-way ANOVA testing for main effects of treatment and time; interaction p<0.001. (J) ORO accumulation after 10 days of primary ASC differentiation; cells treated as in (i). Graph represents ORO absorbance on day 10. Scale=200µm. (K) undifferentiated primary human subcutaneous adipose stromal cells treated with EtOH (Veh), E 2 , 4-OH tamoxifen (TAM) or E 2 +TAM for 7.5 days. Two-way ANOVA testing for main effects of treatment or time; interaction p<0.001. Inset graph is cell number on the final day of proliferation; E 2 versus Veh p=0.011; E 2 +TAM vs E 2 p<0.001 by t-test. Images represent cells at day 7.5 of treatment. (L) ORO in human primary adipose stromal cells. (M) Representative FACS plots of mouse primary ASCs treated with EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM) for 48 hours and stained for Sca1 and CD24. (N-O) Percent of cells positive for Sca1 and CD24 (progenitors; n) or Sca1-positive, CD24-negative (preadipocytes, o) after treated as described in (m). T-tests determined significance.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Staining, Expressing, Proliferation Assay

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A-B) Primary mouse ASCs were treated with EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM) prior to and during adipogenic differentiation for 10 days. (a). Schematic of adipogenesis was created with BioRender https://BioRender.com/az4hymd . Cells were then analyzed for Sca1+/CD24+ progenitors or Sca1+/CD24-preadipocytes by FACS (b). (C) Primary mouse subcutaneous ASCs were isolated from 6 different adult females (ages 20-24 weeks), and plated in media containing EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM). Cells were grown under these conditions and passaged each time they reached 80% confluence. The number of passages was recorded. For each treatment group, the experiment ended when cells no longer reached 80% confluence after 21 days and did not adhere to the plate with the subsequent passage, having reached the end of their proliferative lifespan. The passage number at this point was recorded for each experiment. At each passage, an aliquot of cells was plated for adipogenic differentiation and stained with oil red O (ORO). The absorbance of ORO was measured in technical triplicate wells from cells under each treatment at each passage. (D) Final passage numbers of primary ASCs were recorded under each treatment condition. Mann-Whitney test determined significance. (E) Representative quantification of ORO absorbance from key passage numbers, representing the final passage achieved for each treatment condition (E 2 +TAM=P6; Vehicle=P8; E 2 =P10). Images are phase contrast and ORO-stained plates from the respective passages.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Isolation, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Venn diagram of genes that were higher in cells from obese versus lean female mice treated with E 2 , compared with genes that were lower after treatment of obese females with E 2 +tamoxifen (E 2 +TAM) or estrogen deprivation/withdrawal (EWD). Wisp2/Ccn5 was one of two genes in the overlap of all three gene lists. (B) UMAP showing the distribution and level of Wisp2/Ccn5 expression in primary mouse subcutaneous ASCs. (C) Violin plot showing the level of Wisp2 expression in each cluster, previously defined in Scalzo et al. (D) Bubble plot indicating the average expression level and percent positive cells for Wisp2 in each cluster and treatment group from primary mouse subcutaneous ASCs, as described in Scalzo, et al. (E-G) The percent of Wisp2 positive cells by treatment group in transitional progenitors (e), preadipocytes (f), and adipocyte regulatory cells (AREGs, g).

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Expressing

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Expression of Wisp2 gene (left) and protein (right) in mouse immortalized APCs expressing shRNA for GFP (Con) or Wisp2 (W2KO). (B-D) Expression of adipocyte progenitor markers Cd34 (b), Pdgfra (c), or Cd24a (d) in Con and W2KO immortalized APCs during proliferation in standard growth media. (E) Cell number over 4 days in Con or W2KO populations treated with EtOH (veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM). 3-way ANOVA tested for main effects of time, treatment, and Wisp2 modification. (F) Oil red O (ORO) staining of Con or W2KO cells at day 0 of differentiation. Representative images on the left, quantification of ORO absorbance on the right. (G) Heatmap of expression of progenitor and preadipocyte markers in Con and W2KO cells treated with EtOH (veh) or E 2 for 48 hours. Data are expressed as fold change versus vehicle for each gene, showing 3 replicates per group. (H-I) Quantification of percent Sca1+/CD24+ progenitors (h) and Sca1+/CD24-preadipocytes (i) in Con or W2KO cells treated with vehicle or E 2 . T-tests determined significance.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Expressing, shRNA, Modification, Staining

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Proliferation of mouse primary ASCs during 3 days of treatment EtOH (Veh), E 2 , E 2 +4-OH tamoxifen (E 2 +TAM), exogenous Wisp2 protein (Wisp2), or E 2 +TAM+Wisp2. Two-way ANOVA tested for main effects of time and treatment; interaction p<0.001. Inset shows relative cell number at the final time point. T-test determined significance. (B) oil red O accumulation after 10 days of differentiation of cells treated as in (a). T-tests determined significance at day 10. (C-E) Representative FACS plots showing cells treated as in (a) for 48 hours and stained for Sca1 and CD24. Percent of Sca1+/CD24+ progenitors shown in (d); percent of Sca1+/CD24-preadipocytes shown in (e). T-tests determined significance. (F-K) Expression of Cd34 (f), Pdgfra (g), Sca1/Ly6a (h), Cd24a (i), Pparg (j), or Fabp4 (k) in mouse primary ASCs treated as in (a) and differentiated for 10 days. Cells were treated for the duration of the differentiation assay, replenishing media every 2 days.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Staining, Expressing, Differentiation Assay

Journal: bioRxiv

Article Title: Tamoxifen Targets Wisp2 to Impair Subcutaneous Adipocyte Progenitor Self-Renewal and Adipogenic Differentiation

doi: 10.64898/2025.12.21.695772

Figure Lengend Snippet: (A) Expression of Wisp2 gene (left) and protein (right) in primary mouse subcutaneous ASCs from wild type (Con) or Wisp2 transgenic females (W2OE) both treated with Cre adenovirus in vitro. (B) Oil red O (ORO) accumulation in control and W2OE primary ASCs differentiated for 10 days in vitro. Graph shows ORO absorbance. (C) Proliferation of control or W2OE primary ASCs over 5 days, during treatment with EtOH (Veh), E 2 , or E 2 +4-OH tamoxifen (E 2 +TAM). Three-way ANOVA tested for main effects of time, treatment, or cell genotype. Interaction p-value <0.001. (D) Quantification of cell number after 5 days of proliferation as shown in (c). Statistical analysis is two-way ANOVA testing for main effects of treatment or cell genotype, with post-hoc analyses. (E-F) Quantification of Sca1+/CD24+ progenitors (e) or Sca1+/CD24-preadipocytes (f) after 2 days of treatment with EtOH (Veh), E 2 , or E 2 +TAM measured by FACS. (G) ORO accumulation in CON or W2OE cells treated with EtOH (Veh), E 2 , or E 2 +TAM for 10 days during differentiation. Graph represents quantification of ORO absorbance at day 10. T-tests determined significance.

Article Snippet: Primary human subcutaneous preadipocytes (PCS-210-01, ATCC) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast low serum growth supplement (ATCC PCS-201-041).

Techniques: Expressing, Transgenic Assay, In Vitro, Control