Fig 1: FATE1 and the E3 ligase RNF183 impact the stability of the apoptotic effector BIK.(a) Interaction data for FATE1 based on yeast two-hybrid proteomics analyses. (b) Sixteen hours after transfection in HEK293T cells, lysates (pH 8.0) were incubated with myc antibodies, washed in NDLB and immunoblotted with indicated antibodies. Data representative of three independent assays. (c) WCLs were collected 48 (HCT116) or 72 h (H1155 and HeLa) following siRNA transfection and immunoblotted as indicated. +QVD, cells were exposed to 10 µM of the pan-caspase inhibitor Q-VD-OPh for the duration of the experiment. Data representative of at least two independent assays. (d) HCT116 cells were transfected for 24 h with siCTRL or siBIK then transfected with siCTRL or siFATE for an additional 48 h. WCLs were collected and immunoblotted as indicated. Data representative of three independent assays. (e) Twenty hours post transfection with BIK-L61G, FATE and HA-RNF183 cDNAs, HEK293T cells were lysed in NDLB, immunoprecipitated and immunoblotted as indicated. Data representative of two independent assays. (f) WCLs from HCT116 cells transfected with indicated siRNAs for 72 h were immunoblotted with indicated antibodies. Data representative of two independent assays. (g) WCLs from H1299 cells transfected with indicated cDNAs for 24 h were immunoblotted as indicated. WT, wild-type. CC/AA, C13A/C59A. Data representative of three independent assays. (h) Cell lines from Fig. 2b were transfected for 120 h with siFATE (y-axis) or siRNF183 (x-axis) and cell viability was measured via CTG. Dots represent mean of four independent assays. (i) Viability assays was performed 96 h after siRNA transfection in indicated cell lines. ‘#' indicates independent siRNA pools. Bars represent mean (n=3)±s.d. (j) Kaplan–Meier (KM) survival curves from TCGA colorectal adenocarcinoma patients. HR and P value calculated by Cox Regression Analysis. (k) KM survival curves of patients from GSE42127 as a function of high FATE1- and RNF183-expressing tumours. Venn diagram represents proportion of patients in each group. HR and P value calculated by Cox Regression Analysis. (l) as in k with patients from GSE8894.
Fig 2: FATE1 supports tumour cell viability.(a) Distribution of siFATE1 viability ratios versus all other siCTAs in testbed cell lines with detectable FATE1 expression. Points represent mean of at least two independent assays. P value calculated by Kolmogorov–Smirnov test. (b) Viability assay in indicated cell lines 120 h post siRNA transfection. Bars represent mean viability relative to siCTRL (n=4)±s.d. B, breast; C, cervical; CR, colorectal; L, lung; S, osteosarcoma; O, ovarian; P, prostate; R, renal. (c) Whole cell lysates (WCLs) from indicated cell lines transfected with siCTRL or siFATE for 48 (HCT116), 72 (SUM159, SUM149, H1155) or 96 h (HeLa, HCC366) were immunoblotted with indicated antibodies. Data representative of a minimum of two independent assays. (d) Colony formation assays were performed 48 (HCT116) or 72 h after siRNA transfection. Data representative of two independent assays. (e) WCLs from indicated cell lines were immunoblotted as indicated 96 h after siRNA transfection. Data representative of two independent assays. (f) HeLa cells transfected with indicated cDNAs for 24 h were fixed, immunostained with indicated antibodies (top) and imaged with confocal microscopy. WT, wild-type; ?TM, transmembrane deletion; ?CC, coiled-coiled deletion. Scale bars, 10 µm. Data representative of two independent assays. TOM20 was used to visualize the mitochondria. (g) Top: 48 h after siRNA transfection, WCLs from indicated HCT116 cells (double knockout (DKO)) were immunoblotted with indicated antibodies. Data representative of two independent assays. Bottom: HCT116 cells were transfected and exposed to 10 µM Q-VD-OPh for 48 h, fixed and immunostained for cytochrome c and TOM20. Cytoplasmic cytochrome c was quantitated manually for =200 cells per experiment for each condition. Bars represent mean (n=3)±s.d. Cl, cleaved PARP1; FL, full-length PARP1. (h) WCLs from HCT116 or HeLa cells stably expressing a control construct (HCT116, empty vector; HeLa, pLPCX-GFP) or myc-Bcl-xL were immunoblotted with indicated antibodies 48 (HCT116) or 72 h (HeLa) post siRNA transfection. (i) H1299 cells stably expressing empty vector or myc-FATE1 were exposed to vehicle or 1 µM staurosporine for 6 h. WCLs were immunoblotted with indicated antibodies. Data representative of three independent assays.
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