Mcl-1 regulates the self-renewal of SP cell.

<p>(<b>A, B</b>) Relative expression of Mcl-1 was examined at mRNA and protein levels for indicated cell lines by RT-PCR and western blot analysis. (<b>C</b>) Structure of Obataoclax and its treatment schedule is represented. (<b>D</b>) H1650-SP cells were sorted and plated for self-renewal assay in the presence or absence of Obatoclax at indicated concentration. Average number of spheres generated per well from 1000 cells is plotted (mean±SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. (<b>E</b>) SP cells were sorted from erlotinib resistant H1975 cell line and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated per well from 1000 cells is plotted (mean±SD) and (<b>F</b>) phase contrast microscopy images of the spheres in presence or absence of drugs are presented. (<b>G</b>) Western-blot analysis of βArr1 and βArr2 in Obatoclax treated cells. Inhibition of Mcl-1 did not affect the expression of βArr1 and βArr2 in any of the cell lines tested.</p

NSCLC-SP cells have cancer stem cell-like properties <i>in vitro</i>.

<p>(<b>A</b>) Growth curve on 10,000 H1650-SP or MP cells were generated using MTT cell proliferation assay in regular growth medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055982#pone.0055982-Singh1" target="_blank">[22]</a>. (<b>B</b>) SP or MP cells from H1650 cell line were plated in serum free medium supplemented with EGF and bFGF for 10 days. The average (±SD) number of spheres from 1000 cells is plotted. (<b>C</b>) H1650-SP cells resulted in larger and more colonies compared to MP cells when plated at the density of 1000 cells per 60 mm plate. (<b>D</b>) Cell viability was assayed after 21 days of plating via MTT assay. (<b>E</b>) SP or MP cells were plated on Matrigel and grown in endothelial growth medium (Lonza) overnight. SP cells gave rise to angiogenic like tubules effectively. (<b>F</b>) Real time qPCR analysis on SP and MP cells performed for <i>CD31</i> mRNA expression. (<b>G</b>) CD31 expression on angiogenic tubules as visualized by immunofluorescence.</p

βArr1 regulates the self-renewal of SP cell.

<p>(<b>A</b>) H1650, H1975 and A549 cells were transfected with siRNA against βArr1 and βArr2. Frequency of SP cells was compared with non-targeting control siRNA transfected cells. SP cells are enclosed within the area demarcated in pink. Bar diagrams represent the fold difference in frequency in siRNA transfected cells in respective cell lines. (<b>B</b>) SP frequency was analyzed and represented for A549 cells ectopically expressing rat-βArr1 or GFP and shRNA against βArr1 or non-targeting control shRNA. (<b>C</b>) Real-time PCR and (<b>D</b>) western-blot analysis for ABCG2 expression was performed for these stable cell lines. (<b>E, F, G</b>) A549 cells stably expressing shRNA against βArr1 and non-targeting shRNA were plated for self-renewal assay. (<b>E</b>) Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. (<b>F, G</b>) Average number and size of the spheres generated per well from 1000 cells is plotted (mean±SD).</p

Characterization of SP cells by flow cytometry.

<p>(<b>A</b>) FACS analysis on single cell suspension of human NSCLC cell lines stained with Hoechst 33342 dye showing SP cells. SP cells are enclosed within the area demarcated in pink. Fumitremorgin C inhibited the efflux of the dye and caused the disappearance of SP cells. The frequency of SP cells is represented. (<b>B, C</b>) Cell cycle analysis using area-histogram parameter for the blue emission of Hoechst 33342 as described in the text. The profile shown in B is the representative for all the four cell lines. (<b>D</b>) Aldefluor assay on Hoechst 33342 stained H1975 and H1650 cells. The base line fluorescence was established by inhibiting ALDH activity with DEAB (Left) to generate the gate to identify ALDH-Hi cells in total as well as the SP and MP cells that have not been incubated with DEAB (three right panels). All ALDH-Hi cells are enclosed within the area demarcated in pink. The fold difference in frequency of ALDH-Hi cells between SP and MP cells is plotted.</p