Journal: bioRxiv

Article Title: Endometrial Hyperplasia Risk Is Increased by High-Fat Diet Via Estrogen-Driven Stromal Fibroblast Reprogramming Toward a Pro-Fibrotic State

doi: 10.64898/2026.03.20.713224

Figure Lengend Snippet: (a,b) Representative H&E stain of endometrial tissue from Pten heterozygous mutants for each diet. Main image= 10x magnification; inset=20x magnification; scalebar=300 μm. Caliper indicates the height of an abnormal endometrial gland. (c,d) Representative KRT8 IHC stain of endometrial tissue from Pten heterozygous mutants for each diet (CD n=5; HFD n=4). Main image= 10x magnification; inset=20x magnification; scalebar=300 μm. (e,f) Representative PTEN IHC stain of endometrial tissue from Pten heterozygous mutants for each diet (CD n=9; HFD n=11). Main image= 20x magnification; inset=40x magnification; scalebar=200 μm. Arrow indicates a gland with PTEN loss, while an arrowhead indicates a PTEN intact gland. (g,h) Representative Masson’s Trichrome stain of endometrial stromal ECM from Pten heterozygous mutants for each diet. Main image= 20x magnification; inset=40x magnification; scalebar=200 μm. Arrow indicates endometrial stroma. (i-m) Graphs quantifying EH disease severity in either CD- (n=10) or HFD- (n=11) treated non-estrous staged Pten heterozygous mutant mice from H&E-stained endometrial slides. Measured disease severity parameters were total number of glands per representative histological section, percent normal glands (%), percent abnormal glands (%), average abnormal gland epithelial height (μm), and average abnormal gland area (μm). Datapoints represent a value for each mouse (bar=mean ± s.d.). Two-tailed unpaired t -test statistic with Welch’s correction as needed. (n-q) Graphs quantifying ECM raw integrated density or fold change in integrated density in Pten heterozygous mutant endometrial stroma using Image J with the Color Deconvolution plugin. Samples were from either estrus-staged or non-estrous staged mice in both CD- (estrus n=5; non-estrus n=10) or HFD- (estrus n=4; non-estrus n=11) treated animals. Measurement (datapoint) represents each mouse’s mean of 4 samplings (bar=mean ± s.d.). Two-tailed unpaired t -test statistic. (r) Percent (%) of CD45 + cells from all live single cells in the uterus of CD (n=5) and HFD (n=8) Pten heterozygous mice. Measurement (datapoint) represents each mouse (bar=mean ± s.d.). Two-tailed unpaired t -test statistic. (s) Percent (%) of F4/80 + cells from all CD45 + live single cells in the uterus. Measurement (datapoint) represents each mouse (bar=mean ± s.d.). Two-tailed unpaired t -test statistic with Welch’s correction. (t,u) Percent (%) of dead cells or CD45 + immune cells from all single cells in blood of CD (n=5) and HFD (n=7) Pten heterozygous mice. Measurement (datapoint) represents each mouse (bar=mean ± s.d.). Two-tailed unpaired t -test statistic. (v) Percent (%) of F4/80 + cells from CD45 + single cells in the blood. Blood F4/80 + cells are likely monocytes, as macrophages are not commonly seen in circulation. Measurement (datapoint) represents each mouse (bar=mean ± s.d.). Two-tailed unpaired t -test statistic. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Article Snippet: Samples were labelled 1:50 CD45 (Miltenyi, cat#130-123-784, RRID:AB_2802059), 1:50 Propidium Iodide (PI) (BD Biosciences, cat#556463, RRID:AB_2869075), 1:50 F4/80 (BioLegend, cat#123120, RRID:AB_893479), 1:20 CD80 (BioLegend, cat#104713, RRID:AB_313134), 1:40 CD206 (BioLegend, cat#141707, RRID:AB_10896057), as well as isotype controls at a matching concentration including APC Rat IgG2b,τχ (BioLegend, cat#403806, RRID:AB_ 3096358), Alexa Fluor® 488 Rat IgG2a, κ (BioLegend, cat#400525, RRID:AB_2864283), APC Armenian Hamster IgG (BioLegend, cat#400911, RRID:AB_2905474), and APC Rat IgG2a, κ (BioLegend, cat#400511, RRID:AB_2814702).

Techniques: Staining, Mutagenesis, Two Tailed Test