Fig. 5.

Effects of airway smooth muscle cells and collagen III in colony formation. a Schematic illustrating the overall scheme of conditioned media collection from airway smooth muscle cells (ASMCs) for the evaluation of its impact on colony formation in vitro, and subsequent molecular analysis. b Quantification and size measurement of colonies formed from control and CD167a-overexpressing T24 cancer cells, and c a human metastatic PDX model in a classical soft-agar assay, in the presence or absence of ASMC conditioned media over a 14-day period. Representative phase-contrast images depict the relative colony size from respective conditions. d Quantitative real-time qPCR analysis of COL3A1 mRNA in cultured human ASMCs. Human cDNA mix and cultured T24 cells were harvested as controls. GAPDH was used as reference and the relative expression was normalized against human cDNA mix. e Western blot analysis of COL3 expression in human ASMCs and T24 cancer cells. DNA-PKcs was used as the loading control. f Targeted mass spectrometry analysis to evaluate protein peptides specific toward COL3, that were secreted into the supernatant or conditioned media collected from cultured human ASMCs at 0 h and 16 h after incubation. The amount of COL3 was calculated based on MS1 signal area under curve using SKYLINE software based on top four strongly detected peptides. g Colony formation assay of control and CD167a-overexpressing T24 cancer cells upon COL3 treatment (50 μg ml⁻¹). Number and size of colonies are shown on the left and right panel, respectively. h Colony formation assay of the human metastatic PDX model upon COL3 treatment (50 μg ml⁻¹). Statistical analysis: a two-tailed, unpaired student’s t-test. Error bar: mean ± SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.005