Fig 2: ARHGDIB acts as a downstream effector of KDM6A to mediate the inhibition of cell migration and invasion. a and b siRNA mediated ARHGDIB knockdown in T24 cells was verified by Western blot (a) and qPCR (b), siNC was set as 1. c-e Migration and invasion capacities of the indicated cells were determined by wound-healing assays (c), Transwell migration assays (d), and Transwell invasion assays (e). f Kaplan-Meier survival curve for overall survival of the patients with BCa stratified by ARHGDIB expression levels from tissue microarray (Cat No. HBlaU066Su01; ARHGDIB-low, n = 16, ARHGDIB-high, n = 29). g The protein levels of KDM6A and ARHGDIB in indicated cells were assessed by Western blot. h The mRNA levels of KDM6A and ARHGDIB in indicated cells were detected by qPCR, Puro + siNC was set as 1, Puro + siNC vs KDM6A + siNC, Puro + siARHGDIB vs KDM6A + siARHGDIB. i and j Wound healing assays (i) and Transwell invasion assays (j) of the effects of ARHGDIB knockdown on cell migration and invasion. k and l Cell migration assay of bone marrow monocyte-derived macrophages cocultured with conditioned medium (CM) from indicated cells. m The correlation between KDM6A and ARHGDIB mRNA levels in 12 human fresh BCa tissues was assessed by Spearman’s correlation analysis. n The correlation between KDM6A and ARHGDIB protein levels in tissue microarray (Cat No. HBlaU066Su01) was assessed. o Representative IHC images of KDM6A and ARHGDIB in tissue microarray were shown, Scale bar, 500 μm. All quantification analyses were based on independent triplicate experiments. Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS no significant, based on Student’s t test. Kaplan-Meier analysis based on log-rank test

Fig 3: The role of KDM6A in proliferation of BCa cells. a The protein expression levels of KDM6A in the BCa cells were determined by Western blot. b The lentivirus mediated KDM6A overexpression in BCa cells were examined by Western blot. c and d Effect of KDM6A overexpression on cell proliferation was examined by MTT assays (c) and colony formation assays (d) in the BCa cells. e The protein expression levels of KDM6A in the indicated cells were determined by Western blot. f The image of subcutaneous tumours developed by the indicated T24 cells peeled from mice was shown. g The representative IHC images of KDM6A and Ki67 in subcutaneous tumours were shown. Scale bars, 50 μm (left) and 20 μm (right). h Cell migration assay of bone marrow monocyte derived-macrophages cocultured with conditioned medium (CM) from indicated cells. All quantification analyses were based on independent triplicate experiments. Error bars represent SD. **p < 0.01, NS no significant, based on Student’s t test

Fig 4: KDM6A inhibits BCa cell migration and invasion. a-f Migration and invasion capacities of indicated cells were examined by wound-healing assays (a, d), Transwell migration assays (b, e), and Transwell invasion assays (c, f). The normalized wound area of control cells at 0 h was set as 1. g Representative image of filopodia staining in indicated cells were shown. Scale bars, 10 μm. h Quantification of the number of filopodia per cell was shown. All quantification analyses were based on independent triplicate experiments. Error bars represent SD. i and j Lung metastasis assays of indicated T24 cells. Representative gross lung images of indicated group were shown (left). The number of tumours per lung were counted (right, Puro vs KDM6A, n = 4, NC vs shKDM6A, n = 5). Error bars represent SEM. k Representative HE staining images of the lungs were shown. Scale bars, 200 μm. l Kaplan-Meier survival curve for overall survival of patients with BCa stratified by KDM6A expression levels from tissue microarray (Cat No. HBlaU066Su01; KDM6A-low, n = 23, KDM6A-high, n = 22). *p < 0.05, **P < 0.01, ***p < 0.001, NS no significant, based on Student’s t test. Kaplan-Meier analysis based on log-rank test

Fig 5: Kdm6a‐dependent Kras‐induced oncogenesisH&E staining of pancreata from Kdm6a pKO;Kras G12D and Kras G12D mice. A–CSeven‐day‐old Kras G12D and Kdm6a pKO;Kras G12D mice showed normal morphology.D–IAt 21 days, Kras G12D mice showed normal pancreas morphology (dotted area in D is shown at large magnification in G), whereas Kdm6a pKO;Kras G12D mice already show acinar‐to‐ductal metaplasia (dashed encircled area in F), spindle cell proliferation (asterisks in E and H), sarcomatoid architecture (I), and desmoplastic reaction (black arrowhead in H).J–O Kras G12D mice present occasional acinar‐to‐ductal metaplasia and low‐grade PanINs at 8 weeks (M is a magnification of squared dotted box in J and see Fig 1N), whereas at the same age pancreas from Kdm6a pKO ;Kras G12D mice show massive remodeling (K), extensive acinar‐to‐ductal metaplasia (L and dashed encircled area in N), cancer with prominent spindle/mesenchymal proliferation, infiltrative growth (black arrows and asterisk, respectively, in O) and abundant stroma (black arrowheads in N).Data information: Scale bars: 100 μm (B, D, E, J, K) or 20 μm (A, C, F–I, L–N).