LamB1 translation after heat shock or intervention with ribosome scanning through 2A protease-dependent cleavage of eIF4G

<p><b>Copyright information:</b></p><p>Taken from "The leader region of Laminin B1 mRNA confers cap-independent translation"</p><p></p><p>Nucleic Acids Research 2007;35(8):2473-2482.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885646.</p><p>© 2007 The Author(s)</p> () LamB1 expression in MIM-R cells 4, 6 and 8 h after heat shock as detected by western blotting. () Firefly luciferase assay of MIM-R hepatocytes transfected either with pR-EMCV-F or pR-Lam-F bicistronic plasmids. Cells were exposed to heat shock 12 h post-transfection. 48 hours after transfection, Firefly luciferase activity was determined and normalized to the RNA level after reverse transcription and quantitation of cDNA. () Western blot analysis of MIM-R cells showing the cleavage of eIF4GI/II. MIM-R cells were transfected with wild-type 2A protease expressing plasmid (p2Awt) and lysed at the indicated times. () Firefly luciferase assay of MIM-R hepatocytes co-transfected with pR-F and either p2Amut or p2Awt. () Firefly luciferase assay of MIM-R cells co-transfected either with pR-EMCV-F or pR-Lam-F and p2Amut or p2Awt, respectively. Cells were lysed 48 h post-transfection and the Firefly luciferase activity was normalized to the RNA level after reverse transcription and quantitation of cDNA

MOESM1 of Three-dimensional tumor spheroids for in vitro analysis of bacteria as gene delivery vectors in tumor therapy

Additional file 1: Figure S1. Characterisation of HT-29 MCTS

High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts

<div><p>We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the “cytokine fingerprints” identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.</p></div

The HGF-cMET axis is recapitulated in the Calu-1 FB co-culture model and induces single cell invasion.

<p>(A) and (B) Proteome analysis of cell lysates and supernatants derived either from mono-cultures (Calu-1, NCI-H1437 (H1437), HDF) or from a 2D co-culture (Calu-1+HDF, H1437+HDF). All samples were taken from 24 h cultures as triplicates. For detection of ERK1(T202/Y204), ERK2(T185/Y187) and CREB(S133) the Phospho-MAPK Array (R&D) was used, for AXL(Y779) and MET(Y1234/Y1235) the Phospho-Receptor Tyrosine Kinase Array (R&D), lysis buffer served as control. For HGF the RayBio Cytokine Antibody Array was used, McCoy’s medium plus 10% FCS served as control. (C) MET Western blot analyses utilizing whole cell lysates from mono-cultures (Calu-1, HDF) and from corresponding 2D co-cultures (Calu-1+HDF). Calu-1 and HDF mono-culture lysates were artificially mixed (Calu-1+HDF Mix) to serve as control. (D) p-MET Western blot analyses of whole cell lysates from Calu-1 mono-cultures prior starved in OPTI-MEM for 6 h. Cells were harvested after 15 min of incubation in supernatants (SN) from Calu-1 or HDFs. OPTI-MEM and McCoy‘s 10% FCS medium served as a control. (E) Addition of either recombinant human HGF (rhHGF; 10 ng/ml; left panel) or rhHGF plus anti-hHGF antibody (1 μg/ml; right panel) in a 3D assay (n = 3). The number of invasive single cells (ISC) is depicted on the right for 24 h and 48 h of incubation (n = 3) (Student’s t-test: *p<0.05) (F) Calu-1 spheroids cultured with HDFs (left panel) or with 10 ng/ml recombinant human HGF (rhHGF, right panel). The upper panel shows treatment with 1 μM MET inhibitor crizotinib, whereas the lower panel serves as control (n = 3). Number of ISC is shown on the right. Statistical analysis was performed on the means of ISC by unpaired comparison with Calu-1+HDF (DMSO) or Calu-1+rhHGF (DMSO) samples using Student’s t-test (**p<0.01, ***p<0.001). Scale bar = 100 μM. (G) Calu-1 cells were starved in OPTI-MEM for 6 h and then incubated with HDF conditioned medium (HDF SN) alone or together with 1 μM of crizotinib (HDF SN+crizotinib 1μM). Cells were harvested after 15 min of incubation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. p-MET = Phospho-MET (Y1234/Y1235).</p

Transcription network analysis (Ingenuity) with genes specifically upregulated in co-cultures.

<p>(A) Transcription network based on genes upregulated in all four co-cultures of the non-invasive lung cancer cell line NCI-H1437. (B) Same as in A, but for co-cultures of the invasive lung cancer cell line Calu-1. Genes marked in red were found to be upregulated in co-cultures. FBs used: HDFs, WI-38, NF1 or CAF1, respectively. Selection parameters were: FC>1.5 and p<0.01. (C) Venn-diagram of all differentially regulated NFκB-linked genes (indicated by numbers) found at least once in a co-culture of the invasive Calu-1 or the non-invasive NCI-H1437 cell line with HDF, WI-38, NF1 and CAF1 (FC<-1.5 or FC>1.5 and p<0.01). Gene symbols indicate those genes exhibiting a cytokine activity (AmiGO2 gene ontology platform genes; Term: cytokine activity, Ontology source: molecular_function, Accession: GO:0005125, User filters: taxon_closure_label: Homo sapiens). Out of the seven genes shown in the center of the diagram two (OLR1 and SPP1) are upregulated and five (CASP1, CXCL1, LEF1, PLAU and TFPI2) are downregulated in NCI-H1437 co-cultures and <i>vice versa</i> in Calu-1 co-cultures.</p

Growth behavior, ratio of E-cadherin <i>vs</i>. N-cadherin expression levels, and EMT score of three NSCLC cell line pairs and the CSF2 levels in supernatants of the respective mono- and co-cultures.

<p>(A) E- <i>vs</i>. N-cadherin log-2-transformed relative expression levels (Affymetrix HGU133 Plus 2). (B) Light microscopy pictures of three-dimensional spheroid cultures. Tumor spheroid aggregates were embedded into collagen I and pictures were taken after 72 h. NCI-H1437, NCI-H460 and A549 spheroids showed a non-invasive phenotype (left), whereas Calu-1, NCI-H157 and NCI-H226 spheroids revealed an invasive phenotype (right). Scale bar = 100 μm (n = 3). (C) EMT-score based on EMT signature gene set of Taube <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124283#pone.0124283.ref028" target="_blank">28</a>] and Eddy <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124283#pone.0124283.ref027" target="_blank">27</a>]. A set of EMT signature genes was used to rank invasive (black bars) and non-invasive (grey bars) cell lines according to their EMT score. (D) Levels of CSF2 [pg/ml] in supernatants of mono- (green) and co-cultures (purple: Calu-1 and orange: NCI-H1437). Experiment was performed in triplicates (n = 3). Invasive and non-invasive NSCLC cell lines have been co-cultured with HDFs. Mixes of supernatants of mono-cultures (blue) derived from the respective tumor cell lines and HDFs, respectively, served as controls. Statistical analysis was performed on the mean values by unpaired comparison using Student’s t-test (*p<0.05, ***p<0.001, ****p<0.0001; n.s.: not significant).</p

Percentage of NFκB-related target genes within all differentially regulated genes in a co-culture of Calu-1 or NCI-H1437 with HDF, WI-38, NF1 or CAF1.

<p>Selection parameters were: FC≥1.5 or FC<-1.5 and p≤0.01.</p><p>Percentage of NFκB-related target genes within all differentially regulated genes in a co-culture of Calu-1 or NCI-H1437 with HDF, WI-38, NF1 or CAF1.</p

RT-qPCR for a set of cytokines (CSF2, IL6, IL8 and IL1B) and chemokines (CXCL1 and CXCL6) of total RNA samples derived from mono- and co-cultures in a transwell assay.

<p>The respective co- (+) or mono- (-) culture is indicated on the X-axis. The respective analyzed cytokine or chemokine is indicated in the header of each graph. Expression values are shown in arbitrary units (AU) and have been normalized to beta-2 microglobulin (B2M) mRNA copies. Experiments were performed in triplicates (n = 3). Statistical analysis was performed on the mean values by unpaired comparison of mono-cultured HDF and co-cultured HDF RNA samples by using Student’s t-test (**p<0.01, ***p<0.001; n.s.: not significant).</p

Ingenuity Canonical Pathway Analysis.

<p>The top ranked Ingenuity canonical pathways resulting from differentially upregulated gene expression analysis (FC≥1.5 and p≤0.005) of the respective co-cultures were compared with each other. Calculation of significance was done by Fisher's exact test right-tailed. Canonical pathways exhibiting significant changes in at least three out of the four co-cultures are depicted. Numbers represent—log(p-value).</p><p>Ingenuity Canonical Pathway Analysis.</p

Multi-gene reporter array assay in co-cultures of reporter-construct-transfected FBs.

<p>The different FBs were either co-cultured with themselves or with NCI-H1437 or Calu-1 tumor cells. For each co-culture three independent experiments were performed. (A) Overview (heat map) on Luciferase signals of each of the 45 reporter constructs in transfected FBs (HDF, NF1 and CAF1). Reporters are indicated at the top. Culture conditions are depicted at the right side. Clustering method: UPGMA; distance measure: euclidean; ordering weight: average value. Red arrows indicate the six most significantly upregulated signaling pathways upon co-cultivation which are shown in detail in B. (B) Underlined FBs on the X-axis indicate the cell line which had been transfected with the corresponding reporter construct depicted in the header of each diagram. In all cultures either the same number of transfected FBs were co-cultured with the same number of non-transfected FBs (HDF+HDF, NF1+NF1 and CAF1+CAF1) or accordingly, with the same number of cells of non-transfected tumor cell line (HDF+Calu-1, HDF+H1437 etc.). Controls are depicted in green, Calu-1 co-cultures in purple and NCI-H1437 co-cultures in orange. Statistical analysis was performed by unpaired comparison of control samples (HDF+HDF, NF1+NF1, CAF1+CAF1) with respective co-culture samples by using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001; n.s.: not significant). LI = luminous intensity.</p