Journal: Scientific Reports

Article Title: MicroRNA-21 in cancer-associated fibroblasts supports lung adenocarcinoma progression

doi: 10.1038/s41598-018-27128-3

Figure Lengend Snippet: TGF-β1 induces myofibroblast differentiation and activates miR-21 expression in lung fibroblasts. ( a ) Comparative expression of miR-21 in A549 lung cancer cells, IMR-90 and MRC-5 lung fibroblast cells, and their conditioned media (CM, mean ± SD, n = 3). ( b ) Morphological changes following the stimulation of lung fibroblasts with TGF-β1 for 24 h. ( c ) MiR-21 expression in lung fibroblasts treated with CM from lung cancer A549 cells or with TGF-β1, with and without A83-01 (A83-01 + CM; mean ± SD, n = 3, *** P < 0.001, **** P < 0.0001).

Article Snippet: CM from A549, IMR-90, and MRC-5 cells was produced by culturing cells in serum-free DMEM or MEM for 48 h. For TGF-β1 treatment, IMR-90 or MRC-5 cells were cultured in serum-free medium for one day, followed by the addition of TGF-β1 (R&D Systems, Minneapolis, MN, USA) at 5 ng/mL for 24 h. The concentration of TGF-β1 in A549 cell CM was analysed using the Human TGF-β1 Biotin-Free Immunoassay kit (AlphaLISA, Perkin Elmer, Inc., Waltham, MA, USA) according to the recommended protocol.

Techniques: Expressing

Journal: Scientific Reports

Article Title: MicroRNA-21 in cancer-associated fibroblasts supports lung adenocarcinoma progression

doi: 10.1038/s41598-018-27128-3

Figure Lengend Snippet: MiR-21 up-regulation in lung fibroblasts induces a novel CAF marker, calumenin. IMR-90 or MRC-5 lung fibroblasts transfected with miR-21 mimic (miR-21) or miRNA mimic negative control (NC) were collected for RT-qPCR, western blot, and immunofluorescence analyses. ( a ) IMR-90 or MRC-5 cells were treated with TGF-β1 for 24 h and the expression of CAF markers was evaluated by RT-qPCR (upper panels). Expression levels of CAF markers were examined by RT-qPCR in IMR-90 cells expressing the miRNA mimic negative control (IMR-90 NC ) or miR-21 mimic (IMR-90 miR-21 ), and in MRC-5 cells expressing the miRNA mimic negative control (MRC-5 NC ) or miR-21 mimic (MRC-5 miR-21 ; lower panels, mean ± SD, n = 3). ( b ) Western blot analysis of CAF markers in IMR-90 NC and IMR-90 miR-21 cells (left) and in MRC-5 NC and MRC5 miR-21 cells (right). Cropped images from the same blots are shown. Full-length blots are presented in Supplementary Fig. . Densitometry was performed and the signal intensities were normalized to Actin and expressed as fold change as compared to NC samples. ( c ) Immunofluorescent staining of miR-21-induced calumenin expression in IMR-90 and MRC-5 lung fibroblasts. Calumenin staining is shown in green. Images were acquired at a magnification of 200×. ( d ) Calumenin is expressed in cancer cells and in miR-21-expressing stromal cells in lung adenocarcinoma. Arrowheads indicate the miR-21-positive stromal area. MiR-21 ISH (left) and immunohistochemistry for calumenin (right) in lung adenocarcinomas from the same FFPE block are shown. Scale bars: 100 μm.

Article Snippet: CM from A549, IMR-90, and MRC-5 cells was produced by culturing cells in serum-free DMEM or MEM for 48 h. For TGF-β1 treatment, IMR-90 or MRC-5 cells were cultured in serum-free medium for one day, followed by the addition of TGF-β1 (R&D Systems, Minneapolis, MN, USA) at 5 ng/mL for 24 h. The concentration of TGF-β1 in A549 cell CM was analysed using the Human TGF-β1 Biotin-Free Immunoassay kit (AlphaLISA, Perkin Elmer, Inc., Waltham, MA, USA) according to the recommended protocol.

Techniques: Marker, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Expressing, Staining, Immunohistochemistry, Blocking Assay

Journal: Scientific Reports

Article Title: MicroRNA-21 in cancer-associated fibroblasts supports lung adenocarcinoma progression

doi: 10.1038/s41598-018-27128-3

Figure Lengend Snippet: Calumenin up-regulation in lung fibroblasts: TGF-β1 induction, miR-21-mediated regulation, and effects on lung cancer cells. (a ) Relative calumenin levels (normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) in A549, IMR-90 or MRC-5 cells were evaluated by RT-qPCR (upper panel, mean ± SD, n = 2). Relative calumenin levels (normalised to GAPDH) in IMR-90 or MRC-5 cells treated with CM from lung cancer A549 cells without (CM (A549)) or with the A83-01 TGF-β receptor inhibitor (A83-01 + CM (A549)) or with miR21 inhibitor (miR21 inh + CM (A549)), compared to the normal condition (NC), were evaluated by RT-qPCR (middle and lower panels, mean ± SD, n = 3). ( b ) Relative calumenin levels (normalised to GAPDH) in miR-21-overexpressing IMR-90 or MRC-5 cells treated with TGF-β1 with and without A83-01 treatment were evaluated by RT-qPCR (mean ± SD, n = 3). ( c ) Cell proliferation assay of A549 cells following calumenin siRNA transfection (left, siCALU) or treatment with CM from IMR-90/calumenin siRNA (middle, siCALU) or MRC5/CALU siRNA (right, siCALU) fibroblasts (mean ± SD, n = 3, **** P < 0.0001). Calumenin knockdown efficiency was quantitatively evaluated by RT-qPCR (upper panels). The proliferative capacity of lung cancer cells, cultured in CM from fibroblasts transfected with calumenin siRNA, was decreased compared with the control group, whereas there was no effect on A549 cells transfected with calumenin siRNA (lower panels). ( d ) Migration assay of A549 cells using the IncuCyte ® ZOOM live cell imager following treatment with CM from IMR-90/calumenin siRNA (left, siCALU) or MRC5/calumenin siRNA (right, siCALU) fibroblasts. The data were expressed as the area occupied by A549 cells on the bottom surface, normalised to the initial top value (mean ± SD, n = 4).

Article Snippet: CM from A549, IMR-90, and MRC-5 cells was produced by culturing cells in serum-free DMEM or MEM for 48 h. For TGF-β1 treatment, IMR-90 or MRC-5 cells were cultured in serum-free medium for one day, followed by the addition of TGF-β1 (R&D Systems, Minneapolis, MN, USA) at 5 ng/mL for 24 h. The concentration of TGF-β1 in A549 cell CM was analysed using the Human TGF-β1 Biotin-Free Immunoassay kit (AlphaLISA, Perkin Elmer, Inc., Waltham, MA, USA) according to the recommended protocol.

Techniques: Quantitative RT-PCR, Proliferation Assay, Transfection, Cell Culture, Migration

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: A CD8 T Cell/Indoleamine 2,3-Dioxygenase Axis Is Required for Mesenchymal Stem Cell Suppression of Human Systemic Lupus Erythematosus

doi: 10.1002/art.38674

Figure Lengend Snippet: Lupus CD8+ T cell–derived interferon-γ (IFNγ) promotes indoleamine 2,3-dioxygenase (IDO) production. A and B, Levels of IFNγ in supernatant are increased in the presence of UC-MSCs and SLE CD8+ T cells (A), while no change in levels of transforming growth factor β (TGFβ) is observed (B). Symbols represent individual subjects; horizontal lines show the mean (n = 7 per group). C, CD8+ T cells derived from patients with SLE produce much higher levels of intracellular IFNγ as compared to other cell subsets. D, Recombinant anti-human IFNγ antibody significantly abrogates SLE CD+ T cell–mediated IDO mRNA expression in UC-MSCs. E, Kynurenine levels in supernatant decrease in the presence of anti-human IFNγ antibody. F–H, In cocultured lupus peripheral blood mononuclear cells (PBMCs) and UC-MSCs, anti-human IFNγ antibodies (10 μg/ml) significantly inhibit kynurenine levels in supernatant (F), while the level of tryptophan is increased (G), and the ratio of kynurenine to tryptophan is decreased (H) (effects similar to those observed when 1-methyl- dl -tryptophan [1-DL-MT] is added). I, In the coculture system described above, UC-MSC–mediated inhibition of CD4+ T cell proliferation is abrogated by the addition of anti-human IFNγ antibody. Bars in C–I show the mean ± SEM (n = 7 per group in C, 8 per group in D, 8 per group in E, 5 per group in F–H, and 5 per group in I). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by one-way ANOVA followed by Bonferroni test. PE = phycoerythrin; FITC = fluorescein isothiocyanate; APC = allophycocyanin (see for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38674/abstract .

Article Snippet: The human TGFβ1 ELISA kit was from BioLegend.

Techniques: Derivative Assay, Recombinant, Expressing, Inhibition

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: A CD8 T Cell/Indoleamine 2,3-Dioxygenase Axis Is Required for Mesenchymal Stem Cell Suppression of Human Systemic Lupus Erythematosus

doi: 10.1002/art.38674

Figure Lengend Snippet: Defective indoleamine 2,3-dioxygenase (IDO) production in lupus bone marrow–derived MSCs (BM-MSCs). A–C, Expression of IFNGR1 (A), JAK-2 (B), and STAT-1 (C) was examined by real-time quantitative polymerase chain reaction. D, STAT-1, STAT-3, STAT-5, and their phosphorylated forms were assessed by Western blot analysis after treatment with UC-MSCs alone, UC-MSCs with different cell subsets (CD4+, CD8+, and non-CD4/CD8 T cells), or recombinant human interferon-γ (IFNγ). E, IDO mRNA expression in UC-MSCs, BM-MSCs from healthy controls, and BM-MSCs from SLE patients was examined after stimulation with different cytokines for 48 hours. F and G, Peripheral blood CD8+ T cells derived from patients with lupus were purified and used to stimulate UC-MSCs and BM-MSCs from healthy controls or lupus patients. Forty-eight hours later, IDO gene expression (F) and kynurenine enzyme activity (G) were determined. H, The ability of BM-MSCs from healthy controls and lupus patients to inhibit CD4+ T cell proliferation was compared. Bars in A–C and E–H show the mean ± SEM (n = 7 per group in A–D, 6 per group in E, 3 per group in F and G, and 4 per group in H). ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by one-way ANOVA. TGFβ = transforming growth factor β; IL-6 = interleukin-6; PBMC = peripheral blood mononuclear cell; NS = not significant (see for other definitions).

Article Snippet: The human TGFβ1 ELISA kit was from BioLegend.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Recombinant, Purification, Gene Expression, Activity Assay