Journal: Nature Communications

Article Title: Id1 suppresses anti-tumour immune responses and promotes tumour progression by impairing myeloid cell maturation

doi: 10.1038/ncomms7840

Figure Lengend Snippet: ( a ) Id1 mRNA relative expression levels in day 6 Lin − cells differentiated in the presence of 100 ng μl −1 of murine recombinant proteins (VEGF, IL-6, BMP-7, -9 and -10, and TGFβ compared with Lin − cells differentiated in control media, as determined by qPCR analysis (means±s.e.m., n =6, analysis of variance (ANOVA), **** P <0.0001, ** P <0.01, * P <0.05). ( b ) Id1 mRNA expression levels in day 6 Lin − cells differentiated in the presence of B16F10 CM (abbreviation introduced in Fig 2) alone, with anti-TGFβ and anti-IgG compared with control media, as determined by qPCR analysis (means±s.e.m., n =6, ANOVA, *** P <0.001, ** P <0.01). ( c ) Irf8 mRNA relative expression levels of day 6 WT Lin − cells differentiated in the presence of 100 ng μl −1 of TGFβ, B16F10 CM alone, with anti-TGFβ and anti-IgG compared with control media, as determined by qPCR analysis (means±s.e.m., n =6, ANOVA, **** P <0.0001, *** P <0.001). ( d ) Irf8 mRNA relative expression levels of day 6 Id1 −/− Lin − cells differentiated in the presence of 100 ng μl −1 of TGFβ, B16F10 CM alone, with anti-TGFβ and anti-IgG compared with control media, as determined by qPCR analysis (means±s.e.m., n =6, ANOVA; NS, not significant). Four independent experiments were performed.

Article Snippet: Plasma levels of IFNγ, IL-10 and TGFβ were determined using the Mouse IFNγ and IL-10 Quantikine ELISA Kits and Human TGFβ1 Quantikine Elisa (R&D Systems,) according to manufacturer's instructions.

Techniques: Expressing, Recombinant

Journal: Nature Communications

Article Title: Id1 suppresses anti-tumour immune responses and promotes tumour progression by impairing myeloid cell maturation

doi: 10.1038/ncomms7840

Figure Lengend Snippet: ( a ) qPCR analysis of ID1 (unpaired t -test, * P <0.05), ( b ) S100A8 (unpaired t -test, ** P <0.01) and ( c ) S100A9 (unpaired t -test, ** P <0.01) mRNA expression levels following isolation of CD11B + PBMCs from metastatic melanoma patient blood samples ( n =15) compared with healthy matched controls ( n =7). ( d ) VEGFR1 mean fluorescence intensity levels in CD11B + CD14 − HLA − CD33 + cells PBMCs from metastatic melanoma patient blood samples compared with healthy matched controls, determined by flow cytometry (unpaired t -test, *** P <0.001). ( e ) TGFβ plasma levels from metastatic melanoma patients compared with controls, measured by ELISA (unpaired t -test, ** P <0.01).

Article Snippet: Plasma levels of IFNγ, IL-10 and TGFβ were determined using the Mouse IFNγ and IL-10 Quantikine ELISA Kits and Human TGFβ1 Quantikine Elisa (R&D Systems,) according to manufacturer's instructions.

Techniques: Expressing, Isolation, Fluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Autocrine Sonic Hedgehog Attenuates Inflammation in Cerulein-Induced Acute Pancreatitis in Mice via Upregulation of IL-10

doi: 10.1371/journal.pone.0044121

Figure Lengend Snippet: ( A ) . Morphology of primary acinar cells isolated from mouse pancreas. Magnification, 400×; red scale, 20 µm. ( B ) RT-PCR showed the expression of Il-10 in mice acinar cells isolated from four different mice. Real time PCR showed the expression of Il-10 in mice primary acinar cells treated with ( C ) different concentration of cerulein (0∼20 µM) and ( D ) different concentration of recombinant Shh (0∼0.8 µg/ml). Gapdh was used as an endogenous control (n = 4).

Article Snippet: Briefly, a NUNC 96-well plate (Thermo Scientific, MA) was coated with 5 µg/ml of anti-Shh (Sigma Aldrich, MO) or anti-IL-10 (R&D, MN) capture antibody at 4°C overnight, washed with 0.05% PBST and blocked with 5% bovine serum albumin at room temperature for 1 hours, and then incubated with culture supernatants at room temperature for 2 hours.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Recombinant

Journal: PLoS ONE

Article Title: Autocrine Sonic Hedgehog Attenuates Inflammation in Cerulein-Induced Acute Pancreatitis in Mice via Upregulation of IL-10

doi: 10.1371/journal.pone.0044121

Figure Lengend Snippet: The IL-10 cytokine level in the peripheral blood of mice during the development of cerulein-induced acute pancreatitis was detected by ELISA at ( A ) time points of 0, 8, 16, and 24 hours, and ( B ) in the groups of PBS, cerulein, cerulein combined with IgG control or anti-Shh monoclonal antibody treated for 24 hours. (C) Real time PCR showed that the mRNA level of Il-10 was upregulated by recombinant Shh N-terminal in a dose dependent manner in AR42J cells. (D) The cytokine level of IL-10 in the culture media of AR42J cells treated with recombinant Shh N-terminal was also detected by ELISA. **, p<0.001.

Article Snippet: Briefly, a NUNC 96-well plate (Thermo Scientific, MA) was coated with 5 µg/ml of anti-Shh (Sigma Aldrich, MO) or anti-IL-10 (R&D, MN) capture antibody at 4°C overnight, washed with 0.05% PBST and blocked with 5% bovine serum albumin at room temperature for 1 hours, and then incubated with culture supernatants at room temperature for 2 hours.

Techniques: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Recombinant

Journal: PLoS ONE

Article Title: Autocrine Sonic Hedgehog Attenuates Inflammation in Cerulein-Induced Acute Pancreatitis in Mice via Upregulation of IL-10

doi: 10.1371/journal.pone.0044121

Figure Lengend Snippet: ( A ). Real time PCR showed the mRNA levels of Gli1 and Il-10 in pancreatic acinar AR42J cells transfected with vector control, Shh expression plasmid pJT4-Shh and Shh specific shRNA. ( B ). Real time PCR indicated the changes of mRNA level of Gli1 and IL-10 in pancreatic acinar cell AR42J when overexpression of vector control or Shh shRNA plasmid, or treated with DMSO mock or 10 µM of the Gli inhibitor, GANT61. 2µg of total RNA isolated from fresh tissue was used for reverse transcription, and 1µl of cDNA was used for qPCR reaction. The Gapdh was used as the control. ( C ). ELISA assay showed the Shh and IL-10 cytokine level in the culture supernatant of mice acinar carcinoma AR42J cells transfected with vector control, Shh expression plasmid pJT4-Shh and Shh specific shRNA. ( D ). Western blot confirmed the protein level of IL-10 in AR42J cells transfected with vector control, Shh expression plasmid pJT4-Shh or Shh specific shRNA (upper lane) and Gli1 expression plasmid pcDNA-Gli1 or Gli1 specific shRNA (lower lane).The β-actin ware served as control. **, p<0.001.

Article Snippet: Briefly, a NUNC 96-well plate (Thermo Scientific, MA) was coated with 5 µg/ml of anti-Shh (Sigma Aldrich, MO) or anti-IL-10 (R&D, MN) capture antibody at 4°C overnight, washed with 0.05% PBST and blocked with 5% bovine serum albumin at room temperature for 1 hours, and then incubated with culture supernatants at room temperature for 2 hours.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, shRNA, Over Expression, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot