Additional file 1: Figure S1. of Novel method to detect microRNAs using chip-based QuantStudio 3D digital PCR

Review quality of dPCR chip. Representative images shown chips with good quality as data points above threshold or by calls (upper) and chips below the sufficient quality for miRNAs detection (lower). The data points in the plot are color-coded according to the following call types: FAM (blue), VIC (red), FAM + VIC (green) and NOT AMPLIFIED (yellow). (BMP 1250 kb

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Additional file 1: of MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

Figure S1. In vitro characterization of the immortalized CAF154 fibroblasts. The constitutive high levels of hTERT in all the fibroblasts transduced with retroviral particles (examples shown in A) were confirmed. Nevertheless, almost all the fibroblasts stopped growing after a few population doublings (PDs) and underwent senescence (B) with the exception of CAF154-hTERT cells, which expressed high levels of hTERT (A), showed no signs of senescence (B), and proliferated in a continuous fashion in vitro (C). Cumulative PDs were calculated at the end of every passage in relation to the cell number at the first passage. Of note, despite the immortalization process, CAF154-hTERT maintained the capacity to promote the growth of the adjacent cancer cells in co-culture experiments (D). (TIFF 422 kb

Additional file 5: of MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

Figure S5. FGF-1 and FGF-2 levels are not affected by miR-16. Levels of FGF-1 and FGF-2 in the CM of CAF154-hTERT fibroblasts were transfected with control miR-C and miR-16, collected 72 h after the transfection, and analyzed by multiplex analysis. (TIFF 81 kb

Additional file 2: of MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

Figure S2. In vivo characterization of the immortalized CAF154 fibroblasts. To exclude that the ectopic expression of hTERT and the prolonged culturing had affected the capacity of the CAFs to promote tumor engraftment rate, we characterized the pro-tumorigenic properties CAF154-hTERT cells in vivo by co-injecting CAF154-hTERT and A549 cell lines in immunocompromized mice. We found that the ectopic expression of hTERT did not affect the pro-tumorigenic capability of CAFs to promote the tumor take (A), the volume of the subcutaneous nodules (B), and the dissemination of human cells to the lungs (C) compared to the non-transfected counterpart CAF154 cell line. Based on this evidence, we concluded that the immortalization process did not alter the pro-tumorigenic features of CAF154 cells both in vitro and in vivo. (TIFF 166 kb

Additional file 3: of MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

Figure S3. Potential miR-16 target regions in HGF, FGFR-1, and MEK1 mRNA. FGFR-1 3′UTR was mutagenized to delete to potential miR-16-directed region. (TIFF 86 kb

IL-12Rβ2 expression and function in normal bronchial epithelial cells.

<p>4A. IL-12RB2 expression in human primary bronchial epithelial cells, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); three different NBEC cultures (NBEC #1, #2 and #3) are shown. 4B. IL-12Rβ2 surface expression in human NBEC, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched mAb staining. 4C. Short circuit current recordings in normal bronchial epithelial cells. The figure depicts two representative experiments from control (<i>top</i>) and IL-12 treated (<i>bottom</i>) epithelia showing responses to amiloride (10 µM, apical), forskolin (20 µM, apical and basolateral), UTP (100 µM, apical), and CFTR_inh-172 (10 µM, apical). 4D. Cytokine release by human NBEC, as assessed by Bio-Plex Assay. Pooled results from supernatants of three different bronchial epithelial cell suspensions are shown. IL-12 treatment reduced significantly the release of IL-6 (P = 0.0049), IL-8 (P = 0.0071), FGF-b (P = 0.0231), GM-CSF (P = 0.0028), IP-10 (P<0.0001), MCP-1 (P = 0.0002) and RANTES (P = 0.0108).</p

Anti-tumor activity of IL-12 on NSCLC <i>in vivo.</i>

<p>3A. Volume of tumors grown after Calu6/β2 cell inoculation orthotopically (left panel) or subcutaneously (right panel) in PBS and hrIL-12 treated animals. Animals injected orthotopically were sacrificed after 23 days, those injected subcutaneously after 14 days. Volume of tumors grown after inoculation orthotopically (left panel) or subcutaneously (right panel) of Calu6 cell transfected with the empty vector hrIL-12 treated animals was also shown (empty vector+IL12). The differences in size between tumors removed from PBS and hrIL-12 treated mice were evaluated by Mann-Whitney U test. Boxes indicate values between the 25^th and 75^th percentiles, whisker lines represent highest and lowest values for each group. Horizontal lines represent median values. 3B. Tumors (developed after s.c injection) injected subcutaneously with Calu6/β2 cells in PBS-treated SCID/NOD mice are mostly formed of nests of undifferentiated, pleomorphic and proliferating cells (mitotic (figures) features indicated by arrows) rapidly infiltrating the underlying muscle layers (arrowheads) (a), and supplied by a distinct microvessel network, as assessed by laminin staining (b). In hrIL-12 treated mice, tumor histology is altered by the appearance of large areas of ischemic-hemorrhagic necrosis (N) (c) associated with defective microvascular architecture (d) (×400). Orthotopical injection of Calu6/β2 cells gave rise, in PBS-treated mice, to tumors with istopathological features (e) similar to those of subcutaneously developed tumors (a) and supplied by a well developed microvascular network (f). As observed in subcutaneous tumors, in orthotopic tumors as well hrIL-12 treatment induced wide necrosis (g) and severe microvascular alterations (h) (×400). 3C. Human Angiogenesis PCR Array on tumors explanted from hrIL-12 <i>vs</i> PBS treated animals 23 days after orthotopic inoculation of Calu6/β2 cells. Histogram shows fold expression changes of genes in tumors from hrIL-12 <i>vs</i> PBC treated mice. 3D. Tumors from PBS-treated mice express VEGF-C (a) and IL-6 (c). Expression of VEGF-C and IL-6 is strongly reduced (b and d, respectively) in tumors from hrIL-12 treated mice. (×400).</p

IL-12Rβ2 expression and function in human NSCLC cell lines.

<p>2A. IL-12RB2 expression in NSCLC cell lines, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); PC = positive control (total tonsil B cells); different NSCLC cell lines (Colo699, Calu6, Calu1, A549, SK-MES-1, GLC82 and Calu6/β2 cells) are shown. 2B. Left panel. IL-12Rβ2 protein expression in Calu6/β2 cells, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched antibody staining. Right panel. IL-6 intracellular staining in Calu6/β2 cells cultured with medium or hrIL-12 for 48 h, as assessed by flow cytometry. Open profile: IL-6 staining in cells cultured with medium; dark profile: isotype matched antibody staining, dashed line: IL-6 staining in cells cultured with IL-12. 2C. Angiogenic activity of supernatants from Calu6/β2 cells cultured with medium alone or hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (upper left panel). When supernatants from hrIL-12treated Calu6/β2 cells was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (upper right panel). Lower panels show the angiogenic activity of Calu6/β2 cells in the presence of an anti-IL-6 mAb (left panel) or of an anti-VEGF-C mAb (right panel). These experiments were repeated three times. Original magnification: ×50.</p

IL-12Rβ2 expression and function in human lung adenocarcinoma.

<p>1A. Histological features and IL-12Rβ2 expression in human bronchioloalveolar lung carcinomas. Non-mucinous bronchioloalveolar carcinoma typically shows columnar neoplastic cells growing along the alveolar septa (a). In 41.4% of adenocarcinomas, neoplastic cells lack IL12Rβ2 expression (b), though a few may sometimes retain it (inset in b). By contrast, alveoli unaffected by malignant process express IL-12Rβ2 (c), as observed in the remaining tumors (d). (×400). 1B. Angiogenic activity of supernatants from one representative lung ADC sample cultured in the presence or absence of hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (left panel). When supernatants from hrIL-12 treated lung ADC sample was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (right panel). These experiments were repeated three times. Original magnification: ×50. 1C. Pooled results from human angiogenesis PCR array performed in three lung ADC samples cultured in the presence or absence of hrIL-12 are shown. Histogram shows fold expression changes of genes in primary samples treated with hrIL-12 vs medium.</p