Journal: PLoS ONE

Article Title: C5a Induces the Synthesis of IL-6 and TNF-α in Rat Glomerular Mesangial Cells through MAPK Signaling Pathways

doi: 10.1371/journal.pone.0161867

Figure Lengend Snippet: (A and B) Rat Thy-1N was induced and then the protein level of C5a in the rat renal tissues was detected at different time points (0 h, 0.5 h, 1 h, 2 h and 3 h; n = 4 in each time point) after Thy-1N induction by Western blot assay. (C-E) The mRNA and protein levels of IL-6 and TNF-α in the renal tissues of Thy-1N rats was detected at various time points (0 h, 1 h, 2 h, 4 h, 8 h and 12 h; n = 6 in each time point) after Thy-1N induction by RT-PCR (C and D) and ELISA (E) respectively. Results were represented as means ± SD. Representative photographs were shown. ** P <0.01 versus 0 h time point (non-treated).

Article Snippet: To silence rat C5aR gene, three different siRNA sequences against rat C5aR mRNA (NM053619.1) were designed and synthesized by GenePharma (Shanghai, China).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: C5a Induces the Synthesis of IL-6 and TNF-α in Rat Glomerular Mesangial Cells through MAPK Signaling Pathways

doi: 10.1371/journal.pone.0161867

Figure Lengend Snippet: (A and B) RT-PCR analysis was performed to measure the mRNA levels of IL-6 and TNF-α in rat GMC exposed to C5a (50 ng/ml) for different time points (0 h, 1 h, 2 h, 4 h, 8 h and 12 h). (C) ELISA assay was done to detect IL-6 and TNF-α release from the GMC upon C5a stimulation (50 ng/ml) for above-mentioned time points. (D) ELISA experiments were performed to detect IL-6 and TNF-α release from the GMC stimulated with C5a at the dose of 50 ng/ml or with endotoxin at the dose of 0.001 EU/ml or 50 EU/ml for 8 h. ** P <0.01 versus 0 h time point (non-treated), ns P >0.05 versus MEM group, # P <0.05 versus MEM group. Results were represented as means ± SD ( n = 3 in each time point). Representative photographs were displayed.

Article Snippet: To silence rat C5aR gene, three different siRNA sequences against rat C5aR mRNA (NM053619.1) were designed and synthesized by GenePharma (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: C5a Induces the Synthesis of IL-6 and TNF-α in Rat Glomerular Mesangial Cells through MAPK Signaling Pathways

doi: 10.1371/journal.pone.0161867

Figure Lengend Snippet: (A and B) Western blot assay was performed to determine the expression of C5aR on the GMC incubated with C5a (50 ng/ml) for different time points (0 h, 4 h, 8 h and 12 h). Results were represented as means ± SD ( n = 3 in each time point), and representative photographs were exhibited.

Article Snippet: To silence rat C5aR gene, three different siRNA sequences against rat C5aR mRNA (NM053619.1) were designed and synthesized by GenePharma (Shanghai, China).

Techniques: Western Blot, Expressing, Incubation

Journal: PLoS ONE

Article Title: C5a Induces the Synthesis of IL-6 and TNF-α in Rat Glomerular Mesangial Cells through MAPK Signaling Pathways

doi: 10.1371/journal.pone.0161867

Figure Lengend Snippet: siC5aR was transfected into the GMC to silence C5aR gene followed by C5a stimulation at the dose of 50 ng/ml for different time points, and then above-mentioned molecules were examined by Western blot, RT-PCR and ELISA respectively. (A-D) Western blot was performed to detect the expression of C5aR and the phosphorylation levels of p38 MAPK (A and B), ERK1/2 (A and C) and JNK (A and D) in the GMC after C5a stimulation for 7.5 min (C and D) or 15 min (B). (E and F) RT-PCR was done to determine the mRNA levels of IL-6 and TNF-α in the GMC after C5a stimulation for 4 h. (G) ELISA was used to detect the release of IL-6 and TNF-α from the GMC after C5a stimulation for 8 h. Results were represented as means ± SD ( n = 3 in each group). Representative photographs were shown. ** P <0.01 versus C5a group and siCTR + C5a group.

Article Snippet: To silence rat C5aR gene, three different siRNA sequences against rat C5aR mRNA (NM053619.1) were designed and synthesized by GenePharma (Shanghai, China).

Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Phospho-proteomics

Journal: PLoS ONE

Article Title: C5a Induces the Synthesis of IL-6 and TNF-α in Rat Glomerular Mesangial Cells through MAPK Signaling Pathways

doi: 10.1371/journal.pone.0161867

Figure Lengend Snippet: GMC were incubated with p38 MAPK inhibitor (SB203580, 10 μM), ERK1/2 inhibitor (U0126, 10 μM) and JNK inhibitor (SP600125, 10 μM) respectively for 30 min, and then stimulated with 50 ng/ml C5a for 4 h or 8 h. (A and B) RT-PCR was done to examine the mRNA levels of IL-6 and TNF-α in the GMC at 4 h. (C) ELISA was used to determine the release of IL-6 and TNF-α from the GMC at 8 h. Results were represented as means ± SD ( n = 3 in each group). Representative photographs were exhibited. ** P <0.01 versus C5a group and DMSO + C5a group.

Article Snippet: To silence rat C5aR gene, three different siRNA sequences against rat C5aR mRNA (NM053619.1) were designed and synthesized by GenePharma (Shanghai, China).

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay