Fig. 5: Reverse correlation between MAML1 and Itch in TNBC.

Overall Survival (OS) (A) and Relapse-Free Survival (RFS) (B) Kaplan-Meier curves in Triple Negative Breast Invasive Ductal Carcinoma patients (n = 261) from METABRIC database. Patients were stratified in two groups, High and Low MAML1/Notch1 (left panel) and MAML1/Gli1 (right panel) signature, based on the expression levels of the MAML1/Notch1 and MAML1/Gli1 signature, using the higher tertile as threshold. Statistical significance was assessed using the log-rank test. The pvalues and the number of patients in each group are shown. C Left panel: representative immunohistochemical staining (IHC) of MAML1 expression in primary and metastatic human TNBC, compared to healthy tissue (scale bar: 250 μm or 50 μm, as indicated in the panels). Immunohistochemistry for MAML1 shows different intensities of staining in human TNBC. Right panel: H-scores analysis for MAML1 immunohistochemical staining by one-tailed unpaired t test; *p < 0.05, **p < 0.01. D Representative immunoblot analysis of MAML1 in different TNBC cell lines, compared to non-tumorigenic cell line MCF10a. E In vitro ubiquitination assay of endogenous Itch in MDA-MB-436 and MDA-MB-231, upon small interference of MAML1 (±siMAML1). Immunoprecipitation of Itch with α-Itch antibody and immunoblotting for Ubiquitin to detect the Itch polyubiquitylated forms. α-Itch antibody was used to re-probe blots to assess the levels of immunoprecipitated protein. The lower panel shows the Pre-IP WCE by using α-MAML1 antibody. F PLA analysis by confocal microscope of MDA-MB-436 and MDA-MB-231, upon 48 h of MAML1 silencing (siMAML1), compared to control cells (siScr). Itch ubiquitination levels were detected using a couple of probes α-Itch antibody and α-Ub antibody. Red fluorescence indicates the endogenous levels of Itch ubiquitination. Cell nuclei were stained with DAPI. The right panels show the data, indicated as indicated as intensity mean value (scale bar: 10 μm). G Representative immunoblots for MAML1, Itch, Gli1, Notch1^Val1744, Notch1, Vimentin, N-Cadherin, and PCNA of MDA-MB-436 and MDA-MB-231 upon small interference of MAML1 (±siMAML1). H Left panel: analysis of cell counts in MDA-MB-436 and MDA-MB-231, upon MAML1 silencing (siMAML1), compared to control cells (siScr). Right panel: analysis of MTS absorbance value (490 nm) in MDA-MB-436 and MDA-MB-231, upon MAML1 silencing (siMAML1), compared to control cells (siScr). Data in panel (F and H) represent Mean ± SEM of n = 3 (panel F and left panel H) and n = 5 (right panel H) independent experiments; ***p < 0.001; ****p < 0.0001 calculated by two-tailed unpaired t test. Representative immunoblotting of n = 3 biological replicates with similar results are shown in (D, E, and G). Tubulin was used as loading control. The arrows indicate the molecular weight of the immunoprecipitated protein.