Fig 2: PTEN protein expression in wild-type and DICER mutant HCT116 cells depleted of PTEN ceRNAs.Wild-type (WT) and DICER mutant (DicerEx5) HCT116 cells were transfected with non-targeting control siRNA (siNC) or siRNA plasmids targeting SERINC1 (siSER), VAPA (siVAPA), CNOT6L (siCNO), or PTEN (siPTEN). Cells were harvested 72 hr later for Western blot analysis. (A) Relative protein expression (PTEN/HSP90) are presented for each condition. Western blot bands were quantified, PTEN levels were normalized to HSP90, with protein expression presented relative to siNC. Means reported and error bars represent SD from three independent biological repeats for wild-type HCT116 cells and four repeats for DicerEx5 HCT116 cells. Analysis of wild-type HCT116 cells: one-way ANOVA (equal variance) on PTEN/HSP90 expression: F(4,10) = 25.4, I=3.18×10−5. Planned contrasts between siNC and siSER: t(10) = 1.94, uncorrected I=0.082 with a priori Bonferroni adjusted significance threshold of 0.0125, Bonferroni corrected p=0.326; siNC and siVAPA: t(10) = 5.44, uncorrected p=2.85×10−4, Bonferroni corrected p=0.0011; siNC and siCNOT: t(10) = 3.69, uncorrected p=0.0042, Bonferroni corrected p=0.017; siNC and siPTEN: t(10) = 9.34, uncorrected p=2.97×10−6, Bonferroni corrected p=1.19×10−5. Analysis of DicerEx5 HCT116 cells: one-way ANOVA (unequal variance) on PTEN/HSP90 expression: F(4,6.0) = 19.3, p=0.0014. Planned comparisons: siNC and siSER: two-sample t-test, t(6) = 3.96, uncorrected p=0.0074 with a priori Bonferroni adjusted significance threshold of 0.0125, Bonferroni corrected p=0.030; siNC and siVAPA: two-sample t-test, t(6) = 0.896, uncorrected p=0.405, Bonferroni corrected p>0.99; siNC and siCNOT: Welch’s t-test, t(4.36) = 2.92, uncorrected p=0.039, Bonferroni corrected p=0.156; siNC and siPTEN: two-sample t-test, t(6) = 4.15, uncorrected p=0.0060, Bonferroni corrected p=0.024. (B) Representative Western blots probed with an anti-PTEN antibody and anti-HSP90 antibody. Additional details for this experiment can be found at https://osf.io/drcbw/.

Fig 3: Arid1a deletion promotes endometrial cancer invasion in a Pax8-driven conditional knockout mouse model.a The strategy in generating tissue-specific, conditional Arid1a knockout mouse models. Mice expressing Pax8-driven Cre (Pax8 rTA; TetO-Cre recombinant) were crossed with Arid1aflox/flox (magenta color) or Ptenflox/flox mice (blue color). Flox-mediated gene recombination is activated by Tetracycline (yellow triangle) or its analog, doxycycline, which induces expression of Cre recombinase (orange color) in Pax8-expressing (green color) tissues. Cre subsequently mediates excision of the floxed gene segment of interest. rTA, reverse tetracycline-controlled transactivator; TetO, tetracycline operator; Cre, Cre recombinase; doxy, doxycycline. b Reported ARID1A and PTEN alterations in uterine endometrioid carcinoma and uterine serous carcinoma subtypes of the TCGA uterine endometrial carcinoma PanCancer Atlas dataset. p-values were determined by one-sided Fischer exact test. q-values were determined by Benjamini–Hochberg FDR correction. c Representative images of iAD, iPD, and iPAD uteri 6 weeks after doxycycline treatment. Local and peritoneal disseminated tumors (blue arrows) were only observed in iPAD mice. UT, uterine.

Fig 4: Histopathological characterization of endometrial cancer invasion in Arid1a conditional knockout mouse model.a Diagram summarizing timeline and the corresponding phenotypes of mice with doxycycline-induced gene knockout in the endometrial epithelium. Pathological assessment was not performed on iPAD mice at weeks 16 and 20 due to cancer-related morbidity and mortality. All iPAD control mice were alive and active at both 16 and 20 weeks. Each circle represents an individual uterine sample. d, day; wk, week. iPAD: inducible Pten and Arid1a deletion; iPD: inducible Pten deletion; iAD: inducible Arid1a deletion. b Representative Pax8 immunohistochemistry (expressed in all epithelial cells of mouse uterine epithelium) highlighting the morphology of normal and lesional endometrial epithelial cells of iAD, iPD, and iPAD mice. Mice were treated with doxycycline for the period indicated at the top right corner. Blue star indicates tumor invasion into stroma. Wks, weeks. Scale bars, 300 µm. c Representative H&E (top 2 images) and Pax8 immunoreactivity (bottom 4 images) comparing morphological features between iPAD and iPD carcinomas at 2 weeks and 16 weeks. S, stromal; M, muscle. Scale bars, 200 µm. d Kaplan–Meier survival plot of iPAD mice with (magenta line) or without (black line) doxycycline-induced deletion. The dotted line indicates median survival. ***p < 0.001; log-rank test. e Metastasis of endometrial carcinoma to peritoneal lymph nodes. Left: gross photo from an iPAD mice 4 weeks after doxycycline treatment. Arrows indicate lymph nodes with tumor metastasis. Photomicrographs: H&E stained slides show invasive endometrial carcinoma in the right uterus (asterisk) (upper panel) and metastatic carcinoma within a representative lymph node (bottom panel).

Fig 5: Deficiency of transforming growth factor-β-inducible early gene-1 (TIEG1) decreases infarct size, and alters the expression of molecules in the Pten/Akt and Bcl-2/Bax signalling pathways in myocytes and endothelial cells at the mRNA and protein level. (A) Representative Masson's trichrome staining of the heart to show the infarct zone 28 days post-myocardial infarction (MI). (B) Quantification of infarct zone in heart tissue. #P<0.01 vs. the WT group. (C) Quantitative analysis of mRNA expression of TIEG1, Pten, Akt, Bcl-2, Bax, caspase-3 in cardiomyocytes (CMs). #P<0.01 vs. the WT group. (D) Western blot analysis of the altered expression of Pten/Akt and Bcl-2/Bax signalling pathways in CMs. (E) Quantification of TIEG1, Pten, Akt, Bcl-2, Bax and caspase-3 expession in CMs, n=3. #P<0.01 vs. the WT group. (F) Quantitative analysis of mRNA expression of TIEG1, Pten, Akt, Bcl-2, Bax, caspase-3 in endothelial cells. #P<0.01 vs. the WT group. (G) Western blot analysis of the altered expression of Pten/Akt and Bcl-2/Bax signalling pathways in endothelial cells. (H) Quantification of TIEG1, Pten, Akt, Bcl-2, Bax and caspase-3 expession in endothelial cells, n=3. #P<0.01 vs. the WT group.