Figure 1.

Establishment of long-term acidosis lung cancer cell model. (A) Treatment scheme and analytical approach for long-term acidosis in metastatic colonization. Patient-derived CLS1 lung cancer cells were acclimated for 2 months in pH 7.4 or pH 6.6 media. Both in vitro and in vivo assays were validated by clinicopathological examination of NSCLC specimens. (B) Time lapse microscopy across 96 h to observe doubling dynamics and morphology at a magnification of x40. Each color bar indicates one doubling generation, from generation 0 (blue) to 4 (red). (C) Average doubling time of cell generations (G0-G4) presented as the mean ± SD from three independent experiments. (D) Viability of pH 7.4 and pH 6.6 cells as examined using trypan blue exclusion assay at 0, 24, 48, 72 and 96 h post-seeding based on three independent experiments. (E) Metabolic curve as determined using MTT assay at 24, 48, 72 and 96 h based on two independent experiments with three replicates each. (F) Protein expression of the stemness-associated transcription factors Oct4 and Nanog. Representative western blot image using 20 µg whole cell lysate per lane. (G) ATP-binding cassette-transporter-dependent clearance activity. Gated areas mark the reserpine-sensitive side population cells. Data were analyzed using Student's unpaired t-test and are presented as the mean ± SD. Mol. Wt, molecular weight; NSCLC, non-small cell lung cancer; OD, optical density; GO, Gene Ontology; PPI, protein-protein interaction; KEGG, Kyoto Encyclopedia of Genes and Genomes.