Anti cd98

We performed cell surface staining with fluorescence-conjugated antibodies in 2% FBS–PBS. We obtained fluorochrome-conjugated anti-CD11b, anti-F4/80, anti-CD71, and anti-CD98 from Biolegend. We identified cell death using a violet Live/Dead kit (Invitrogen). We performed intracellular staining for Alexa Fluor 647-conjugated p-Akt (Ser473) and p-4EBP1 (Thr37/46) (Cell Signaling Technology) using BD Biosciences Cytofix/Cytoperm and Perm/Wash solutions. We collected data using a BD FACSAria II and analyzed them with FlowJo (Treestar).

SUM-159 cells plated on coverslip chambers (Thermo Fisher, Cat. # 155379) were fused at their corresponding time points, fixed with 4% PFA (EMS), and then permeabilized and blocked with blocking solution (0.5% Triton X-100, 10% BSA in PBS) for 1 h. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C. After 3 washes (10 min) with PBS, cells were incubated with Alexa Fluor-conjugated secondary antibodies. The following primary antibodies were used: Anti-Yap1 (Cell Signaling 14074S, 1:250), Anti-p21 (Cell Signaling, Cat. # 2947S, 1:200), Anti-pH3 (anti-Phospho-Histone H3 (Ser10); Cell Signaling, Cat. # 3377, 1:200), Anti-clathrin heavy chain (Abcam, Cat. # ab21679, 1:200), Anti-AP-2 (Abcam, Cat. # ab189995, 1:200), Anti-Glut1 (Abcam, Cat. # ab40084, 1:100), Anti-CD98 (BioLegend, Cat. # 315602, 1:200) and Anti-CD147 (BioLegend, Cat. # 306202, 1:200). For imaging and quantification, at least a total of 15 fields of view were randomly chosen by Hoechst 33342 nuclear staining (Thermo Fisher, Cat. # 62249) and imaged by Zeiss LSM880 confocal or NIKON TIRF microscope. At least three different samples were quantified per treatment type at each respective time point.