Epr25a

ChIP assay was performed according to the instructions of the ChIP assay kit (Millipore, Bedford, MA, USA). HEK293T cells were plated into a 10-cm culture dish and transfected with the pCMV-HA-POU6F1 plasmid for 72 h. Cells were fixed with 1% formaldehyde for 10 min and then quenched with 125 mmol/L glycines. After being washed with cold PBS twice, the cells were collected and sonicated. Afterward, IP was conducted by using antibodies for HA (Abcam, ab9110), POU6F1 (abclonal, A7299), or IgG (Abcam, EPR25A). The co-precipitated DNAs were purified with phenol/chloroform for further PCR and analysis. Primer sets were listed in Supplementary Table 5.

Flow cytometry was used to determine the expression of membrane-associated proteins (MHC class I and MHC class II) in 4T1 cell lines treated with F3 (50 μg/ml), dissolved in 0.1% dimethyl sulphoxide, for 24 h. The dose concentration of F3 was previously shown to inhibit 4T1 cells in vitro in a dose- and time-dependent manner [23 (link)]. The primary antibodies used were as follows: Anti-MHCI (Rabbit monoclonal, EPR1394Y; Abcam, Cambridge, UK); Anti-MHCII (Mouse monoclonal, MRC OX-6; Abcam); Rabbit IgG isotype control (EPR25A, Abcam); Mouse IgG1 isotype control (X092701, Dako Denmark A/S). Untreated cells and isotype controls were used as negative controls. Cellular expression of a specific protein was based on the detection of target antigen bound with fluorescent-labeled antibody at equilibrium. For a successful fluorescence activated cell sorting (FACS) of live cells using the flow cytometer, the calibration procedure, reagent preparations and dilution of antibodies were carefully performed to obtain optimum intensity of the fluorescence signal. Data for 10,000 live events were acquired for each sample using a FACS Calibur cytometer for analysis. The FACS results were analyzed using the FlowJo v.X.0.7 software (Tree Star Inc., Ashland, OR, USA).