Fig 2: Schematic representation for ASH2L acting as a co-activator of ERa to be involved in promotion of endometrial cancer progression

Fig 3: ASH2L associates with MLL1/WDR5 to enhance ERa-induced transactivation. A, Co-IP was performed to detect the interactions between endogenous ERa and ASH2L/MLL1/WDR5 in shCtrl and shASH2L Ishikawa cells. B, Interaction domains of ASH2L with MLL1/WDR5, or ERa in Ishikawa cells was tested by Co-IP. C, Luciferase assay shows the effects induced by deletion of ASH2L on the ERa-mediated transactivation, which is upregulated by WDR5 or MLL1 in Ishikawa cells. Expression of WDR5 and MLL1 was detected using western blotting. *Asterisks placed to indicate the position of expression of FLAG-ASH2L truncated mutants. Data are shown as mean ± SD (n = 3), *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 interacts with ERa in ECa-derived cell lines. A, Endogenous interaction between ASH2L and ERa in Ishikawa cells was verified by co-immunoprecipitation. *Asterisk in (A) on the second band in the ASH2L panels indicates the expression of ASH2L. B, Exogenous association between ASH2L and ERa in Ishikawa cells which are transiently expressing ERa and FLAG-ASH2L plasmids by Co-IP. C, Identification of binding domains in ERa for ASH2L interaction. Bound proteins in GST pull-down experiments were analyzed using immunoblotting using anti-FLAG antibody and equal loading of GST-ERa deletion mutants was assessed by Coomassie brilliant blue staining. Schematic representation of GST-tagged ERa-AF1 (29-180) and ERa-AF2 (282-595) was shown at the top of (C). *Asterisk placed to indicate the expression position of GST or GST-ERa truncated mutation proteins. D, Immunofluorescence confocal experiments were used to identify the localizations of endogenous ERa (red) and ASH2L (green). E, Diagram of full-length (FL) and truncated mutants of ASH2L. F, Fluorescence confocal experiments were used to identify the localizations of exogenous ERa (Red) and ASH2L-FL or its truncations (green). Nucleus was stained using DAPI (blue). Scale bars, 10 µm