Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Indicators of necroptosis were discovered in brain tissues after ICH. ( a ) The necroptosis of cells in brain tissues was detected by PI labeling. As shown, compared with the Sham group, considerable PI+ cells were detected in frozen sections of brain tissues in rats at 24 h after ICH. Arrows point to PI+ cells. Scale bar=50 μ m. ( b ) Related with ( a ), it revealed relative levels of PI+ cells, ** P <0.0001 versus Sham group, unpaired t -test, n =6. ( c ) The results of western blot suggested that the expression levels of RIP1, a major regulator for necroptosis, were obviously upregulated, and it reached peak at 24 h in brain tissues after ICH. ( d ) Related with ( c ), quantitative analysis of expression levels of RIP1 in brain tissues within 1 week after ICH. ** P =0.0003 versus Sham group, unpaired t -test, n =3. ( e ) Double immunofluorescence (IF) analysis was performed with antibodies for RIP1 (green) and NeuN (red). Nuclei were fluorescently labeled with DAPI (4',6-diamidino-2-phenylindole) (blue). Representative images of the Sham group and the ICH (24 h) group were shown. Scale bar=10 μ m. ( f ) The formation of necrosome was detected by immunoprecipitation (IP) by using anti-RIP1 antibody (rabbit immunoglobulin G (IgG) was also used as a negative control), and RIP3, MLKL and caspase-8 were detected by immunoblotting. The results suggested that increased interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were observed in brain tissues at 24 h after ICH. Input, 5% of extract before IP. ( g ) Quantitative analysis of IP. ** P =0.0011 versus Sham group; && P =0.0011 versus Sham group; # P =0.0152 versus Sham group; all were unpaired t -test, n =3. Besides, results of IF ( h ) showed that expressions of these four proteins, which constituted necrosome, were increased significantly in brain tissues at 24 h after ICH than those in the Sham group. Scale bar=100 μ m. Related statistic of data was revealed in ( i ). ** P <0.0001 versus Sham group; && P <0.0001 versus Sham group; ## P <0.0001 versus Sham group; $$ P <0.0001 versus Sham group; all were unpaired t -test, n =6. All data are expressed as means±S.E.M., mean value for the Sham group was normalized to 1.0

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: Labeling, Western Blot, Expressing, Immunofluorescence, Immunoprecipitation, Negative Control

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Different effects of Nec-1 and z-VAD on brain injury after ICH. Nec-1 (the specific inhibitor of necroptosis) and z-VAD (a caspase inhibitor) were used to explore whether necroptosis contributes to brain injury after ICH. ( a ) The necroptosis in cells were detected by PI staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Nec-1 reduced the PI+ cells, whereas it had no significant effects on TUNEL+ cells (apoptosis), and z-VAD only downregulated the TUNEL+ cells. Additionally, combination of Nec-1 and z-VAD obviously both reduced the PI+ cells and TUNEL+ cells. Scale bar=100 μ m. ( b ) Corresponding bar graph revealed relative levels of PI/TUNEL+ cells. ** P <0.0001 (both in PI and TUNEL) versus Sham group; NS, not significant difference ( P =0.0909 in PI and P =0.0857 in TUNEL) versus ICH group; && P <0.0001 in PI and NS ( P =0.4233) in TUNEL versus vehicle group; NS ( P =0.4233) in PI and ## P <0.0001 in TUNEL versus vehicle group; $$ P <0.0001 in PI and NS ( P =0.0663) in TUNEL versus z-VAD group; all were unpaired t -test, n =6. ( c ) IP showed that Nec-1 reduced interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8, indicating that the formation of necrosome was inhibited. Input, 5% of extract before IP. ( d ) Corresponding bar graph revealed relative levels of association. ** P =0.0097 in RIP1, ** P =0.0099 in RIP3, ** P =0.0010 in MLKL and ** P =0.0022 in caspase-8 versus Sham group; NS, not significant difference ( P =0.3077 in RIP1, P =0.7942 in RIP3, P =0.9047 in MLKL and P =0.4930 in caspase-8) versus ICH group; & P =0.0102 in RIP1, & P =0.0493 in RIP3, && P =0.0074 in MLKL and NS ( P =0.9349) in caspase-8 versus vehicle group; NS ( P =0.8658) in RIP1, NS ( P =0.6857) in RIP3, # P =0.0205 in MLKL and # P =0.0281 in caspase-8 versus vehicle group; $$ P =0.0065 in RIP1, $ P =0.0499 in RIP3, $ P =0.0403 in MLKL and NS ( P =0.6746) in caspase-8 versus z-VAD group; all were unpaired t -test, n =3. ( e ) Expression of albumin, which is regarded as the index of BBB injury, was increased after ICH, whereas it could be significantly decreased when treated with Nec-1 and/or z-VAD. ( f ) Corresponding bar graph revealed relative levels of albumin. * P =0.0200 versus Sham group; NS, not significant difference ( P =0.9779) versus ICH group; & P =0.0461 versus vehicle group; # P =0.0496 versus vehicle group; $ P =0.0126 versus z-VAD group; all were unpaired t -test, n =6. ( g ) Compared with ICH group, the brain water content was partially attenuated in Nec-1 and/or z-VAD group, ** P <0.0001 (both in Ipsi-CX and Ipsi-BG) versus Sham group; NS, not significant difference ( P =0.9022 in Ipsi-CX and P =0.9660 in Ipsi-BG) versus ICH group; & P =0.0109 in Ipsi-CX and && P =0.0077 in Ipsi-BG versus vehicle group; # P =0.0162 in Ipsi-CX and NS ( P =0.0727) in Ipsi-BG versus vehicle group; $$ P =0.0015 in Ipsi-CX and $$ P =0.0039 in Ipsi-BG versus z-VAD group; all were unpaired t-test, n =6. ( h ) The levels of TNF- α in the CSF were measured by ELISA and reduced by Nec-1 treatment. ** P <0.0001 versus Sham group, NS, not significant difference ( P =0.7897) versus ICH group; && P <0.0001 versus vehicle group, ## P =0.0008 versus vehicle group, $$ P <0.0001 versus z-VAD group; all were unpaired t -test, n =6. All data are expressed as means±S.E.M. and mean value for Sham group was normalized to 1.0.

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: Staining, TUNEL Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Clinical behavior scores in each group ( n =6)

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques:

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Knockdown of RIP1 reduced necroptosis in brain tissues after ICH. Knockdown and overexpression of RIP1 were used to study the role of RIP1-mediated necroptosis after ICH in rats. ( a ) PI+ cells decreased in the Si-RIP1 group (knockdown), whereas they increased in the Ad-RIP1 group (overexpression). Arrows point to PI+ cells. Scale bar=200 μ m. ( b ) Related with ( a ), it revealed relative levels of PI+ cells. ** P <0.0001 versus Sham group; NS, not significant difference ( P =0.8453) versus ICH group; && P <0.0001 versus Si-NC group; NS, not significant difference ( P =0.9211) versus ICH group; ## P <0.0001 versus Ad-GFP group; all were unpaired t-test, n =6. ( c ) IP demonstrated that interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were significantly inhibited in the RIP1-knockdown group, whereas they increased in the overexpression group. Input, 5% of extract before IP. Quantitative analysis of IP was shown in ( d ), * P =0.0177 in RIP1, * P =0.0261 in RIP3, ** P =0.0072 in MLKL and * P =0.0209 in caspase-8 versus Sham group; NS, not significant difference ( P =0.7939 in RIP1, P =0.6515 in RIP3, P =0.4640 in MLKL and P =0.8812 in caspase-8) versus ICH group; & P =0.0254 in RIP1, & P =0.0184 in RIP3, && P =0.0019 in MLKL and && P =0.0010 in caspase-8 versus Si-NC group; NS ( P =0.7254) in RIP1, NS ( P =0.5590) in RIP3, NS ( P =0.3154) in MLKL and NS ( P =0.6284) in caspase-8 versus ICH group; # P =0.0164 in RIP1, # P =0.0138 in RIP3, # P =0.0367 in MLKL and ## P =0.0083 in caspase-8 versus Ad-GFP group; all were unpaired t -test, n =3. ( e ) Expression of albumin was elevated in the Ad-RIP1 group, whereas it was opposite in the Si-RIP1 group. ( f ) Bar graph related to ( e ). ** P =0.0007 versus Sham group; NS, not significant difference ( P =0.8780) versus ICH group; & P =0.0493 versus Si-NC group; NS, not significant difference ( P =0.9361) versus ICH group; # P =0.0470 versus Ad-GFP group; all were unpaired t -test, n =3. ( g ) Brain water content decreased in the Si-RIP1 group, whereas it was opposite in the Ad-RIP1 group,** P <0.0001 (both in Ipsi-CX and Ipsi-BG) versus Sham group; NS, not significant difference ( P =0.4041 in Ipsi-CX and P =0.5003 in Ipsi-BG) versus ICH group; && P <0.0001 in Ipsi-CX and & P =0.0209 in Ipsi-BG versus Si-NC group; NS, not significant difference ( P =0.5276 in Ipsi-CX and P =0.6015 in Ipsi-BG) versus ICH group; ## P <0.0001 (both in Ipsi-CX and Ipsi-BG) versus Ad-GFP group; all were unpaired t -test, n =6. ( h ) The levels of TNF- α in the CSF were reduced in RIP1-knockdown group, whereas the effect was diametric in the RIP1 overexpression group. ** P <0.0001 versus Sham group; NS, not significant difference ( P =0.5570) versus ICH group; && P =0.0002 versus Si-NC group; NS, not significant difference ( P =0.9151) versus ICH group; ## P <0.0001 versus Ad-GFP group; all were unpaired t-test, n =6. All data are expressed as means±S.E.M., and mean values for Sham group were normalized to 1.0. Ad-GFP, adenovirus with GFP; Ad- RIP1, adenovirus with RIP1; Si-NC, Si-negative control.

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: Knockdown, Over Expression, Expressing, Negative Control

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Conditioned medium-induced necroptosis in neurons in vitro . We used two in vitro ICH models to further explore the role of inflammatory factors, such as TNF- α , in inducing necroptosis: one is neurons treated with OxyHb directly, and the other is using supernatant of culture of microglia (stimulated with OxyHb in advance) as neurons' conditioned medium, and treated with or without inhibitor of TNF- α . ( a ) The necroptosis and apoptosis of neurons in vitro were detected by PI and Annexin V double staining and flow cytometry analysis, respectively. PI−/Annexin V− represented survival neurons, PI+/Annexin V− represented necroptotic neurons, PI−/Annexin V+ represented apoptotic neurons, and PI+/Annexin V+ represented a mixed damage of neurons. The results of flow cytometry indicated a higher ratio (~27.8%) of apoptosis and a lower ratio (~11.4%) of necroptosis when neurons were stimulated with OxyHb. However, in conditioned medium treatment group, it was a higher percentage of necroptosis (~31.5%), whereas the ratio could be significantly reduced when treated with TNF- α inhibitor (~13.5%). ( b ) Related bar graph showed four different conditions of neurons in various groups; NS, not significant difference ( P =0.4642) in PI+/Annexin V− cells and ** P =0.0007 in PI−/Annexin V+ cells versus Control group; && P =0.0001 in PI+/Annexin V− cells and NS, not significant difference ( P =0.1498) in PI−/Annexin V+ cells versus Control group; ## P =0.0004 in PI+/Annexin V− cells and NS, not significant difference ( P =0.3401) in PI−/Annexin V+ cells versus Conditioned medium group; all were unpaired t -test, n =3. ( c ) IP revealed that when treated with conditioned medium, interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were remarkably increased. And these results were attenuated when pretreated with TNF- α inhibitor. ( d ) Consistent data analysis of IP. ** P =0.0047 in p-Ser, ** P <0.0001 in RIP3, ** P <0.0001 in MLKL and ** P =0.0002 in caspase-8 versus Control group; && P <0.0001 in p-Ser, && P =0.0006 in RIP3, && P <0.0001 in MLKL and && P <0.0001 in caspase-8 versus Control group; ## P =0.0003 in p-Ser, ## P =0.0018 in RIP3, ## P =0.0012 in MLKL and ## P =0.0002 in caspase-8 versus conditioned medium group. The mean values for the control group were normalized to 1.0, all were unpaired t -test, n =3. ( e ) Immunofluorescence analysis was performed with antibody for RIP1 /RIP3 (green), MLKL (red) and caspase-8 (purple) in cultured primary neurons under indicated treatment. Nuclei were fluorescently labeled with DAPI (blue). Representative images were shown. Arrows indicated the colocalization of RIP1-MLKL-caspase-8 and RIP3-MLKL-caspase. Scale bar=20 μ m. ( f ) PI and Hoechst double staining was also used in detection of necroptosis. The results showed that neurons in conditioned medium had higher ratio of necroptosis (as arrows point to, PI+/Hoechst+ cells), which could be inhibited by TNF- α inhibitor. Scale bar=50 μ m. ( g ) Numbers of PI+/Hoechst+cells. ** P <0.0001 versus control group, && P <0.0001 versus control group, ## P <0.0001 versus conditioned medium group; all were unpaired t -test, n =6. All data are expressed as means±S.E.M.

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: In Vitro, Double Staining, Flow Cytometry, Control, Immunofluorescence, Cell Culture, Labeling

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Phosphorylation of RIP1 switched necroptosis in cultured neurons in model of ICH in vitro . As phosphorylation of RIP1 has an essential role of necroptosis, we used overexpression and with a mutation of phosphorylation site (S166A) of RIP1 to investigate its role in necroptosis in ICH. ( a ) Flow cytometry indicated that, compared with the conditioned medium group, it turned up a higher ratio of necroptosis (~66.7%) and a lower ratio of apoptosis (~0.9%) in neurons during overexpression of RIP1 by Ad-RIP1 transfection. Besides, this phenomenon could be significantly reduced in the S166A group (necroptosis ~34.2%) and in the Nec-1 treatment group (necroptosis ~21.7%). ( b ) Related bar graph showed four different conditions of neurons that had been stated above. ** P =0.0001 in PI+/Annexin V− cells versus the conditioned medium group; && P =0.0027 in PI+/Annexin V− cells versus the Ad-RIP1 group; $$ P <0.0001 in PI+/Annexin V− cells and $$ P <0.0001 in PI−/Annexin V+ cells versus the Ad-RIP1 group; all were unpaired t-test, n =3. ( c ) IP demonstrated that interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were increased in the Ad-RIP1 group. These increased interactions could be attenuated remarkably in the S166A group. ( d ) Consistent data analysis of IP. ** P =0.0015 in p-Ser, ** P =0.0004 in RIP3, ** P =0.0013 in MLKL and ** P =0.0003 in caspase-8 versus the conditioned medium group; & P =0.0493 in p-Ser, & P =0.0126 in RIP3, && P =0.0036 in MLKL and && P =0.0018 in caspase-8 versus the Ad-RIP1 group. The mean values for the control group were normalized to 1.0; all were unpaired t -test, n =3. ( e ) PI and Hoechst staining indicated that the ratio of necroptosis upregulated (as arrows point to, PI+/Hoechst+ cells) in the overexpression group, whereas mutation of the phosphorylation site inhibited this effect. Scale bar=50 μ m. ( f ) Numbers of PI+/Hoechst+ cells, ** P <0.0001 versus the conditioned medium group; && P <0.0001 versus Ad-RIP1 group; all were unpaired t -test, n =6. All data are expressed as means±S.E.M.

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: Phospho-proteomics, Cell Culture, In Vitro, Over Expression, Mutagenesis, Flow Cytometry, Transfection, Control, Staining

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Hypothesized model for molecular mechanism involved in necroptosis in brain tissues after ICH. After ICH occurs, microglia were rapidly activated and released an amount of inflammatory cytokines such as TNF- α . TNFR1 can spontaneously trimerize at the plasma membrane. When TNF- α binds to TNFR1, the conformation of these receptor trimers would be changed, allowing their cytosolic tails recruit multiple proteins, and then develop a complex (complex I) including TRADD (TNF- α receptor-associated death domain), RIP1, TRAF2 (TNFR associated factor 2), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1/2). Deubiquitylation of RIP1 mediates its transition from complex I to complex II, and then cooperates with RIP3 for recruitment of MLKL (mixed lineage kinase domain-like protein), FADD (FAS-associated protein with a death domain) and caspase-8. In this complex IIb, RIP1 and RIP3 are inhibited by caspase-8. When caspase-8 is inactive, complex IIb would carry out the TNF- α -mediated necroptotic pathway. Further, complex of MLKL and RIP3 transfers to the cell membrane and forms a channel, and then the internal flow of Ca 2+ or Na + is caused. Finally, the cell is dead by the necroptotic pathway. As necroptosis can further promote inflammation, it suggests that it can possibly have a positive feedback relationship between necroptosis and inflammation in brain injury after ICH.

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: Clinical Proteomics, Membrane

Journal: Cell Death & Disease

Article Title: Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

doi: 10.1038/cddis.2017.58

Figure Lengend Snippet: Experimental designs. ( a ) Experiment 1 was designed to show the time course of RIP1 expression after ICH and to determine a time point for the next experiment. ( b ) Experiment 2 was designed to explore the roles of RIP1 and necroptosis in brain injury after ICH in vivo . ( d ) Experiment 3 was designed to study the potential mechanism of RIP1 and necroptosis in brain injury after ICH in vitro.

Article Snippet: The following two kinds of siRNAs were used: (1) disorganizing rat RIP1 mRNA (Si-RIP1) to silence its transcription and (2) scramble siRNA (Si-negative control) (both from Genescript).

Techniques: Expressing, In Vivo, In Vitro

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