Fig 2: Knockdown of ASH2L inhibits tumor growth in mice xenograft tumor. A, Pictures show xenograft tumors formed by Ishikawa cells in mice. B and C, Tumor volume (B) and weight (C) of xenografts in each group were shown. D, RNA levels of ASH2L in each group were detected by qPCR. E and F, Protein levels of ASH2L and PAX2 in every xenograft tumor tissue were detected by western blotting. G and H, Expression of ASH2L and proliferation-related protein Ki67 in the xenograft tumor tissues were examined by immunohistochemistry. Data are shown as mean ± SD. *P < .05, ***P < .001

Fig 3: ASH2L is highly expressed in endometrial cancer. A, Kaplan-Meier analysis for overall survival of 516 endometrial cancer (ECa) patients based on the data from the Cancer Genome Atlas (TCGA). B, Expression of ASH2L in 29 benign endometrial tissues (N) and 48 endometrial cancer (T) detected using western blotting. C, Expression of ASH2L in (B) was quantified using densitometry shown as box and whisker plots. D, Immunohistochemical (IHC) assay in paraffin sections of BET and ECa (histological grades I/II/III) in ×100 magnification (scale bars, 200 µm) and ×400 magnification (scale bars, 50 µm). E and F, Average optical density (AOD) of IHC stained by ASH2L antibody in BET and ECa specimens. Data are shown as mean ± SD, *P < .05, **P < .01, ***P < .001

Fig 4: ASH2L facilitates the recruitment of MLL1 to the cis-regulatory elements of the ERa-regulated gene in endometrial cancer cells. A, Schematic diagram of the searched estrogen response elements (EREs) at cis-regulatory elements of PAX2. B, ChIP assay with indicated antibodies shows the recruitment of ASH2L and ERa at different EREs. C, ChIP assay shows the recruitment of indicated protein and histone modification at EREII/III. DNA fragments were amplified using qPCR with the primers indicated in (A). D and E, ChIP-Seq profiles and the corresponding peaks of genomic regions around PAX2. Error bars represent mean ± SD (n = 3), *P < .05, **P < .01, ***P < .001

Fig 5: ASH2L upregulates ERa-mediated transactivation in ECa cells. A, Luciferase reporter assay was performed to verify the effects of ASH2L-FL or different truncation on the ERa-mediated transcriptional regulation above; western blotting shows the expression of ASH2L-FL and its truncation below. B, Schematic representation of ERa, ERa-AF1, and ERa-AF2 is shown at the top of the panel. Luciferase reporter assays were performed to detect the effects of ASH2L on the transcriptional activity mediated by ERa or its truncated mutants in COS7 cells. Expression of ASH2L was analyzed using western blotting. C, Transcriptional activity of ERa was detected in Ishikawa cells infected with lentivirus carrying shASH2L or shCtrl. Expression of endogenous ASH2L below. D, Real-time qPCR assay was performed to detect the effects of ASH2L on activation of several genes regulated by E2-ERa. ASH2L was knocked down by specific siRNA in Ishikawa cells. E, Western blotting was performed to detect the effects of ASH2L on the expression of PAX2 or Cyclin D1 in Ishikawa cells. F, Western blotting was performed to detect the effects of different doses of ASH2L on the expression of PAX2. Quantification of protein expression is shown as histograms on the right. *Asterisk in the western blotting is placed to indicate the position of labeled proteins. In the histogram, the bars represent the mean ± SD (n = 3), *P < .05, **P < .01, ***P < .001