path4588-sup-0001-FigureS1.tifTIFF image, 306.3 KB FIR over‐expression in NSCLC tissues. (A) Transcriptomic data derived from normal lung tissue (n = 10) and ADC (n = 86) revealed significantly elevated FIR levels (all splice variants) in tumour tissue 17 n = 4; ADC, n = 40; small cell lung cancer (SCLC), n = 4; SCC, n = 13) 18

path4588-sup-0002-FigureS2.tifTIFF image, 428.4 KB Exclusive nuclear expression of FIR in NSCLC cells. (A) Analyses of nuclear and cytoplasmic protein fractions derived from three independent NSCLC cell lines (A549, CaLu‐1 and CaLu‐6), followed by FIR detection of cells; PARP and β‐tubulin served as fractionation controls. (B) Transient transfection of two independent and gene‐specific siRNA (nos 1 and 2) silencing all FIR variants revealed efficient reduction at the protein level compared to the respective controls; ctr, untreated control; Nons, nonsense siRNA; densitometric measurements revealed a reduction of at least 70% in all analysed cell lines (A549, CaLu‐6, CaLu‐1)

path4588-sup-0003-FigureS3.tifTIFF image, 690.4 KB Functional relevance of nuclear FIR in A549 and CaLu‐6 cells. (A) Silencing of all FIR splice variants in A549 and CaLu‐6 cells resulted in a significant reduction of cell viability (MTT assay). (B) FIR inhibition was associated with a strong reduction of NSCLC proliferation in A549 and CaLu‐6 cells (BrdU–ELISA). (C) Western immunoblotting revealed increased cleavage of PARP after FIR silencing in both cell lines analysed. (D) Inhibition of FIR equally induced the numbers of apoptotic cells (FACS analysis). For (A, B, D) the Mann–Whitney U‐test was utilized; nonsense siRNA‐transfected cells (#) were used for statistical comparison

path4588-sup-0004-FigureS4.tifTIFF image, 5.5 MB FIR supports CaLu‐1 migration. (A) Efficient FIR inhibition using independent gene‐specific siRNAs recognizing all splice variants did not affect CaLu‐1 viability. (B) Instead, reduced FIR amounts significantly diminished the ability of CaLu‐1 cells to cover the gap in a culture dish; migration efficiency was measured 18 h after 'scratching' and calculation of cell‐free areas. (C) For the reduction of possible off‐target effects from specific siRNAs and to achieve dosage effects, siRNAs were mixed and transfected in different concentrations (20 and 40 n m ); as expected, 40 n m siRNA led to stronger FIR silencing and inhibition of cell migration (D). For (B) the Mann–Whitney U‐test was utilized

path4588-sup-0005-FigureS5.tifTIFF image, 7.9 MB Comparative analysis of FIR, FBP, SAP155, E‐cadherin and p27Kip1 expression in primary NSCLC tissues. (A) Comparison of FIRtotal expression with FBP, SAP155, E‐cadherin and p27Kip1, using NSCLC TMAs; exemplary stains for tissues with high or low FIR abundance are shown; to illustrate that the p27Kip1 protein was successfully stained, different samples with high FIR levels are shown; bar = 50 µm. (B) Correlation between FIRtotal and FBP or c‐Myc transcript levels in primary NSCLC tissues. For (A, B), Spearman's coefficient is indicated as a measure of association

path4588-sup-0006-FigureS6.tifTIFF image, 596.9 KB Effect of FIR on proteins regulating cell mobility. Inhibition of FIRtotal by gene‐specific siRNA revealed a reduction of RhoB and phospho‐CDC42 concentrations afterwards; RhoA, RhoC, moesin/ezrin/radixin and the respective phospho‐isoforms were not affected. Protein amounts were quantified and normalized to the respective actin concentration (graphs). Densitometric quantification was performed via Quantity One software (Bio‐Rad, Munich, Germany)

path4588-sup-0007-FigureS7.tifTIFF image, 2.2 MB Sequence of image processing. Exemplary image processing illustrating the step‐wise improvement of picture quality and data acquisition: original brightfield, after shade correction, after illumination correction, after denoising, after local thresholding and Sobel edge detection, after application of image morphological operators and removal of artefacts, and final segmented structures in brightfield picture

path4588-sup-0008-FigureS8.tifTIFF image, 830.3 KB Impact of FIRtotal and FIRΔexon2 on potential downstream effectors. (A) Western immunoblot analysis of c‐Myc, E‐cadherin, FBP, p27Kip1 and SAP155 after siRNA‐mediated silencing of FIRtotal and FIRΔexon2 in CaLu‐1 cells. (B) Correlation of FIRΔexon2 transcript levels with FBP mRNA amounts in primary human NSCL tissues. For (B), Spearman's coefficient was calculated as a measure of association

path4588-sup-0009-TableS1.pdfPDF document, 490.1 KB siRNA sequences

path4588-sup-0010-TableS2.pdfPDF document, 186.6 KB Primers for semi‐quantitative real‐time PCR Primers for nested PCR (FIR)

path4588-sup-0011-TableS3.pdfPDF document, 283 KB Primary and secondary antibodies