(A) Intracellular levels of biotinylated Tfn in siRNA-transfected H1299 cells were quantified at the indicated time points after a 30-minute pulse. The percentage of the internalized Tfn pool was calculated relative to the initial loading (n = 4 samples per condition). (B) WB analysis of CD44, ITGB1, and AXL protein levels in WCLs (input) or streptavidin bead–enriched protein samples from H1299 cells that were transfected with PI4K2A/TurboID construct and treated with biotin. Controls included the PI4KB/TurboID construct (PI4KB) and Turbo alone (CTL). (C) WB analysis of WCLs (input) or anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the control IP. (D) SPP1 protein-protein interaction network (STRING-db.org). (E) WB analysis of proteins isolated by streptavidin bead–based pulldowns carried out on H1299 cells treated with biotin-labeled recombinant SPP1. (F) Confocal micrographs of plasma membrane–bound ITGB1 (arrows, upper panels) and CD44 (arrows, lower panels) in nonpermeabilized siRNA-transfected H1299 cells stained with antibodies against endogenous ITGB1 or CD44. Scale bars: 10 μm. (G) WB analysis of cell membrane–enriched fractions (Mem.) and WCL. Densitometric values are shown under the gels. (H) WB analysis of cleaved PARP1 (gel) and flow cytometric analysis of annexin V/PI–stained cells (graph) to quantify apoptosis in siRNA-transfected H1299 cells. (I) Boyden chamber migration and invasion assays on siRNA-transfected cells. (J) Schematic illustration of the working model. PI4K2A coordinates exocytic and endocytic vesicular trafficking to activate an SPP1-dependent autocrine loop. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 1-way ANOVA test for multiple comparisons (A, H, and I).