(A) H1299 cells expressing CLCa and TfnR ligands. Top row, representative WT cell (TfnR ligand, GI = 4.6, LI = 1.6). Bottom row, representative AKT-inhibited cell (TfnR ligand, GI = 6.0, LI = 0.3). Scale bar 10 μm. (B) ARPE-19 cells. Top row, representative WT cell (TfnR ligand, GI = 4.3, LI = 0.8). Bottom row, representative AKT-inhibited cell (TfnR ligand, GI = 4.6, LI = 1.6). (C–D) LI and GI of CLCa-TfnR co-localization for Ctrl (red) and Aktinh. cells (cyan). Every data point represents the LI and GI values for a single cell. Statistical analyses performed with the Wilcoxon rank-sum test. All DeBias analyses were performed with K = 40. (C) H1299: N number of cells Ctrl = 30, Aktinh. = 30; number of CCPs per cell: Ctrl = 455.5, Aktinh. = 179.5. GI p-value < 0.0001, LI p-value < 0.0001. (D) ARPE-19: N number of cells Ctrl = 30, Aktinh. = 20; number of CCPs per cell: Ctrl = 958.8, Aktinh. = 1138.2. GI p-value < 0.002, LI p-value < 0.008. (E–F) Receiver Operating Characteristic (ROC) curves showing the true positive rates as a function of false-positive rates for single cell classification, higher curves correspond to enhanced discrimination (Materials and methods). Black – LI, orange – (GI,LI). Statistics via permutation test (Materials and methods). (E) H1299 AUC: (GI,LI) = 0.96 versus LI = 0.88, p-value ≤ 0.003. (F) ARPE-19 AUC: (GI,LI) = 0.83 versus LI = 0.72, p-value ≤ 0.048. (G–H) Joint distributions of CLCa (x-axis) and TfnR (y-axis) for H1299 (G) and ARPE-19 (H) cells. (I–J) Marginal distributions of CLCa (left) and TfnR (right) for H1299 (I) and ARPE-19 (J) cells. (K–L) Combined CCP lifetime distribution for 50 Ctrl (red) and Aktinh. (cyan) cells. Statistics with Wilcoxon rank-sum test (Materials and methods). (K) H1299: p-value < 0.006 (mean EMD: Ctrl = 29.3 versus Aktinh. = 43.6); number of cells: 50 (Ctrl), 11 (Aktinh.). (L) H1299: p-value n.s. (mean EMD: Ctrl = 36.1 versus Aktinh. = 38.0); number of cells: 12 (Ctrl), 12 (Aktinh.). (M–N) Percentage of TfnR uptake: Ctrl versus Aktinh. (whiskers - standard deviation). Statistics via two-tailed Student’s t-test. (M) H1299: p-value < 0.005; N = 3 independent experiments. (N) ARPE-19: p-value n.s.; N = 3 independent experiments.