Additional file 1: of Characterization, localization and comparison of c-Kit+ lung cells in never smokers and smokers with and without COPD

Figure S1. Absence of cross reactivity between host species in primary and secondary antibodies. Figure S2. Representative image of a lung tissue mosaic. Figure S3. Percentage of c-Kit+/CD45+ gated cells by flow cytometry in the three study groups. Figure S4. C -kit+ cell populations in lung tissue. Figure S5. Representative image showing that C-kitlowCD45- cells determined by IF stain positively for CD31. Figure S6. Representative images showing C-Kit+ cells with stem cells markers. Table S1. Primary and secondary antibodies for immune-histochemistry staining. Table S2. Clinical characteristics of the subpopulaton included in the immunofluorescence analysis (mean ± SD). (ZIP 1150 kb

Overexpression of miR-200c affects cell migration.

<p><b>(A)</b> After 36 or 48h of transfection with pre-miRNAs in the NSCLC cell lines, cell migration was measured by <i>in vitro</i> scratch assay. High levels of miR-200c reduced cell migration in comparison with control in all cell lines (H23, p = 0.005; A-549, p = 0.0085; HCC-44, p = 0.013), while high levels of miR-141 reduced cell migration only in A-549 (p = 0.04). <b>(B)</b> E-cadherin levels were evaluated by immunohistochemistry and increased levels were observed in cells transfected with pre-miR-200c in comparison with those transfected with pre-miR-141 or pre-miR-control in all three cell lines.</p

OS analysis in the entire cohort.

<p>OS according to <b>(A)</b> miR-200c expression levels in the entire cohort (N = 155); <b>(B)</b> miR-200c expression levels in stage I patients (N = 94); and <b>(C)</b> miR-141 expression levels in stage I patients (N = 94).</p

Main patient characteristics.

<p>Main patient characteristics.</p

Multivariate analysis for OS in the entire cohort (N = 155) and in the subgroup of patients with adenocarcinoma (N = 73).

<p>Multivariate analysis for OS in the entire cohort (N = 155) and in the subgroup of patients with adenocarcinoma (N = 73).</p

OS in 73 patients with adenocarcinoma according to the miR-141/miR-200c risk score.

<p>OS in 73 patients with adenocarcinoma according to the miR-141/miR-200c risk score.</p

Concordance between ALK IHC and <i>ALK</i> FISH.

<p><i>IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.</i></p><p>Concordance between ALK IHC and <i>ALK</i> FISH.</p

Box plots for number of <i>ALK</i> positive cells by FISH automatized technique versus intensity of the ALK IHC staining.

<p>With the Ventana anti-ALK antibody (A) and with Novocastra (5A4) antibody (B). Kruskal-Wallis test was performed. The comparisons between the categories in each antibody were statistically significant (p<0,001).</p

Immunostaining pattern of ALK in NSCLC using Ventana anti-ALK (D5F3) and Novocastra (5A4) antibodies.

<p>ALK IHC reveals variable levels of protein expression: from absent (0) to weak/faint cytoplasmic staining (1+) in negative cases and from moderate (2+) to strong (3+) granular cytoplasmic immunstaining in positive tumors. In ALK IHC-negative cases, the immunoreactivity was always 0 by Novocastra (5A4) IHC, whereas ranged from 0 to 1+ by Ventana antibody. However, in ALK IHC-positive cases, protein expression was always 3+ by Ventana antibody, whereas it ranged from 2+ to 3+ by Novocastra (5A4) IHC. Original magnification: 400×.</p

Study design and specimen selection.

<p>Study design and specimen selection.</p