Journal: In Vivo

Article Title: Effect of Decay-accelerating Factor on LPS-induced Acute Lung Injury in the Rat

doi: 10.21873/invivo.14124

Figure Lengend Snippet: Lung tissue complement regulation in wild type (WT) and Daf knock-out (Daf -/- ) rats following lipopolysaccharide (LPS) administration using western blotting. Protein lysates derived from lung tissue samples were obtained from WT and Daf -/- rats following LPS administration (16 h) and analyzed using SDS-page for A) C3b deposition and B) DAF and C5aR1 expression (C). Graphs represent densitometric analysis results for C3b and C5aR over GAPDH used as loading control. Bars represent mean±SEM. Statistical analysis performed using One-way Anova testing for more than two group comparisons. Post-hoc analysis performed using Fisher’s least significant difference test. *p<0.05.

Article Snippet: Antibodies for detection of rat DAF (clone RDIII-7 cat no: HM3035), rat C3/C3b (clone 2B10B9B2, cat no: HM3031), rat Crry (clone TLD-1C11, cat no: HM3032) and rat C5aR (clone mAb R63, cat no: HM3017) were purchased from Hycult (Uden, the Netherlands).

Techniques: Knock-Out, Western Blot, Derivative Assay, SDS Page, Expressing, Control

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

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: A schematic view of the experimental design. Cells were obtained from the hippocampal formation of rat embryos (E18.5) using enzymatic and mechanical dissociation methods. Neuron-astrocyte co-cultures were used to investigate C5aR expression/activation and the effect of C5a on cell viability. The microfluidic device was used to quantify the C5a effect on axonal growth.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Activation Assay

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: Primers used for gene expression by RT-PCR.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: C5aR expression in neuron-astrocyte co-cultures by RT-PCR. ( A ) C5aR mRNA expression was observed in both injured and intact cell cultures. Controls included expression of housekeeping GAPDH mRNA. ( B ) Double immunostaining for NF-L (green) and C5aR (red) indicated that injured and intact neurons expressed NF-L ( a , e ) and C5aR ( b , f ) on their membranes ( n = 3). Merged images in yellow ( c , g ) revealed a co-localization of these markers. Controls for each condition were performed by omitting the primary antibodies ( d , h ) and nuclei were counterstained using DAPI. The dotted lines and * indicate the site of injury. Scale bars: 50 µm. ( C ) Double immunostaining of NF-L in red ( a , e ) and C5aR in green ( b , f ) on spinal cord cryosections. Controls were performed by omitting the primary antibodies ( d , h ) and nuclei were counterstained in blue using DAPI. Images revealed that C5aR was co-expressed with NF-L in grey and white matters (yellow in ( c , g )). Scale bars: 50 µm.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Double Immunostaining

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: Interaction between C5a and neuron C5aR. Cells were cultured in the B-27-supplemented neurobasal medium as a control condition without C5a ( a ), stimulated with C5a (50 ng/mL) ( b ) or with both C5a and PMX53 ( c ) for 5 min ( n = 3). Immunostaining of the phosphorylated C5a receptor (C5aR-P, red) and NF-L (green) indicated that while NF-L was expressed in all conditions, C5aR-P was not expressed by cells when the inhibitor was added. Scale bars: 20 µm.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Cell Culture, Control, Immunostaining

Journal: Cells

Article Title: Complement C5a Implication in Axonal Growth After Injury

doi: 10.3390/cells13201729

Figure Lengend Snippet: Axonal growth in the microfluidic device. ( A ) Cells were incubated with the control medium for 48 h either without C5a ( a , d ), or with C5a (50 ng/mL) ( b , e ), or with both C5a and PMX53 ( c , f ). GFP labeling allowed visualization of the axonal growth via pictures taken at 40×. The axonal growth is reported by combining the images obtained over the 48 h growth period. Scale bars: 100 µm. ( B ) After axonal injury, the axonal length significantly increased under all conditions between 24 h (white) and 48 h (hatching). Adding C5a significantly increased the axonal length at 24 h and at 48 h as compared to the controls at the same delays. This increase was suppressed after adding the C5aR inhibitor. Results are presented as boxplots. The central line in each boxplot represents the median values while the box edges indicate the first and third quartiles. All points beyond the whiskers up to 1.5 times the interquartile range are considered as outliers. # indicates a significant ( p < 0.05) difference between incubation periods for the same treatment. * and + indicate significant ( p < 0.05) differences between the axonal lengths after adding C5a as compared to the control at 24 and 48 h, respectively. $ and & indicate significant ( p < 0.05) differences with C5a + PMX53 compared to the C5a treatment at 24 and 48 h, respectively. Ө indicates significant ( p < 0.05) differences with C5a + PMX53 compared to the control at 24 h.

Article Snippet: Rabbit IgG against phosphorylated rat C5aR (AY-AF8362) was purchased from Euromedex (Souffelweyersheim, France); rabbit IgG anti-rat C5aR (CPA4586) from CliniSciences (Nanterre, France); Alexa Fluor 594 goat anti-rabbit IgG (A11012), Alexa Fluor 488 goat anti-rabbit IgG (A11034), Alexa Fluor 594 donkey anti-mouse IgG (A21203), and rabbit IgG anti-rat NF-L (PA587394) from ThermoFischer Scientific; mouse IgG anti-rat glial Fibrillary Acidic Protein (MAB360) from Merck Millipore (Darmstadt, Germany); and mouse IgG anti-rat oligodendrocyte transcription factor (sc-515947) and mouse IgG anti-rat neuronal Differentiation 2 (sc-365896) from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Incubation, Control, Labeling