(A) A549 cells were incubated with liposomes containing 10 ng SOCS3 or an equal volume of liposomes containing PBS alone (empty) for 72 h and analyzed by CyQuant cell proliferation assay measuring total cellular DNA (n = 3 separate experiments). (B) One thousand A549 cells were plated per well of a 96-well tissue culture plate and were incubated with empty or SOCS3 liposomes for 72 h. Cellular confluence was measured using an IncuCyte Live Cell Analysis system. Data points represent the mean of quadruplicate measurements of confluency, expressed as AU per well, and are representative of triplicate wells per condition. (C) A549 cells were treated with empty or SOCS3 liposomes for 24 h and analyzed for FoxM1 mRNA expression by qPCR (n = 3 separate experiments). (D) Representative micrographs of caspase-3/7–positive cells after liposome treatment. Concurrent with the confluence assay in B, A549 cells were incubated with IncuCyte caspase-3/7 apoptosis assay reagent and empty or SOCS3 liposomes. Fluorescence micrographs are representative of 3 triplicate wells per treatment. Scale bar: 100 μm. (E) A549 cells were treated with empty or SOCS3 liposomes or FasL (as a positive control) for 24 h, and annexin V⁺ cells determined via flow cytometry (n = 4–5 replicate samples). (F) A549 cells were treated with empty or SOCS3 liposomes for 24 h and analyzed for relative FasR mRNA expression by qPCR (n = 4 separate experiments). (G) A549 cells were treated with FasL, empty liposomes, or SOCS3 liposomes for 24 h, with or without the pan-caspase inhibitor Z-VAD-FMK (Z), and cell proliferation was determined by CyQuant assay (n = 5–14 replicates from 3 separate experiments). (H) Top: Schematic depiction of CSE transformation protocol for WT or G12V-RLE cells. A total of 0.5 × 10⁶ RLE-6TN cells were incubated with either WT or G12V retrovirus for 24 h. Cells were washed and selected with puromycin for an additional 48–72 h. Surviving cells were passaged an additional 2 times before use in experiments. For CSE transformation, cells were plated at a concentration of 10,000 cells/well of a 6-well tissue culture dish and treated with 3% CSE, along with empty or SOCS3 liposomes, for 1 h daily for 7 consecutive days. They were then cultured in equal numbers in a soft agar colony assay for an additional 7 days before colonies were enumerated. Bottom left: Representative micrographs of CSE-treated WT or G12V RLEs within a soft agar colony assay. Photographs were taken at 40× magnification. Scale bar: 1000 μm. Bottom right: Data represents mean colony counts from 7 separate 40× fields per treatment from 3 separate experiments. Student’s 2-tailed t test was used for comparison of 2 groups (A, C, F, and H), or 1-way ANOVA with Bonferroni’s post hoc analysis was used for multiple comparisons (E and G); *P < 0.05 or #P < 0.05 represents comparison to control or empty liposomes, respectively.