Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3a in B cells is generated extracellularly by alternative pathway C3-convertases. Western blot results investigating the origin of C3a in Raji B cells. Cells were incubated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37 °C. To analyze complement pathway dependent generation of C3a, sera depleted in C1q (classical pathway), MBL (lectin pathway) or Factor B (alternative pathway) were used. To distinguish intracellular or C3-convertase dependent processing of C3, C3, and C3met were added alone (intracellular cleavage) or in the presence of C3-depleted serum (C3 convertase supplementation). C3 processing and C3a generation were analyzed by Western blot with the goat polyclonal anti-C3 (Quidel) and rabbit anti-C3a (Complement Technologies) antibodies under reducing conditions. Result shown is one representative out of three independent experiments.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Generated, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a enter the nucleus after uptake. (A,B) Western blot results showing presence of C3 and C3a in nuclear compartments of Raji cells. The human B cell line, Raji was incubated with NHS, C3 or C3met as a source of C3 either in EDTA-GVB (A) or in Mg-EGTA (B) buffer for 1 h at 37°C. After lysis, cytoplasmic, soluble nuclear, and chromatin-associated nuclear fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody under non reducing conditions (A) or with the rabbit polyclonal antibody against C3a under reducing condition (B) . The purity of distinct cellular fractions was verified using antibodies against B-actin (cytoplasmic marker), lamin B1 (nuclear marker) and histone H2B (chromatin-associated nuclear marker). Results shown are one representative experiment out of three (A) or four (B) independent analyzes. (C) Representative confocal images showing that AlexaFluor 488 labeled C3 enters the nucleus. 2 × 10 6 Raji cells were incubated with 100 μg/ml C3-AlexaFluor 488 for 30 min at 37°C, fixed and counterstained with DAPI using mounting medium. Representative images are shown from two independent experiments investigating at least 50 cells/analysis.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Western Blot, Incubation, Lysis, Marker, Labeling

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a bind to distinct types of DNA. (A–C) Gel shift assays presenting interaction between C3(a) and genomic DNA isolated from Raji cells. Isolated genomic DNA was incubated either with pre-titrated concentrations of C3 (A) or C3a (B) or C3b (C) and separated by agarose gel electrophoresis. Formation of high molecular weight DNA-protein complexes is indicated by slower DNA migration (DNA shift) compared to migration of the free nucleic acid. The presence of C3 and its cleavage fragments in the pre-formed protein-DNA complexes was confirmed with anti-C3a and anti-C3d antibodies caused supershift. One representative experiment out of five performed is shown. (D) Gel shift assays showing that C3(a)—DNA interaction is not ionic in nature. C3 or C3a were incubated with genomic DNA of Raji cells in the presence of increasing NaCl concentration (ranging from 150 to 1,200 mM). Binding of C3 and C3a to DNA was observed at 8X higher salt concentration than the physiological 150 mM. (E) Interaction of C3 and C3a with DNA is not restricted to genomic DNA. Linearized pCEP4 vector was incubated with either C3 or C3a and DNA-protein complex formation was investigated by gel shift assay. Results illustrated are one representative out of three independent experiments. (F,G) ELISA results confirming interaction between C3(a) and DNA. ELISA microplate surfaces were coated either with DNA (F) or C3, C3a, C3b (G) . After incubation with distinct concentrations of C3 proteins (F) or Raji genomic DNA (G) , protein-DNA interactions were detected using anti-C3d, anti-C3a (anti-C3 ELISA, F ), or anti-dsDNA antibody (anti-DNA ELISA, G ). Results shown are mean absorbance values measured at 490 nm with SD of four independent experiments. (H) ELISA results showing presence of C3-DNA complexes in formaldehyde cross-linked chromatin fraction of Raji B cells. Cells were incubated in the presence or absence of C3, C3met, fixed with 1% formaldehyde to cross-link protein-DNA complexes and chromatin isolated using commercial kit. C3-DNA complexes were investigated by ELISA on anti-C3 or anti-DNA coated plates. Data are shown as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to non-treated cells (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01).

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Electrophoretic Mobility Shift Assay, Isolation, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Migration, Concentration Assay, Binding Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Interaction of C3 and its cleavage fragments with histones. Recombinant histone core octamers (H2A/H2B/H3/H4), the linker H1 histone, or alpha-1-antitrypsin (negative control) were immobilized on microtiter plates and incubated with increasing concentrations of C3 (A) , C3b (B) , or C3a (C) . As negative control, BSA was used. Binding was detected either with monoclonal anti-C3d (A,B) or polyclonal anti-C3a (C) antibodies. Background signals obtained from BSA incubation were subtracted from the original values. Data are presented as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to 0 μg/ml protein incubated surfaces (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Upper asterisks indicate statistical significance of H1-, lower asterisks of histone octamer coated surfaces compared to binding to A1AT. (D) gDNA of Raji cells was coated on microtiter plates and AlexaFluor 488-labeled H1 histone binding was monitored in the presence or absence of C3a. As negative control, BSA was used. Background signals obtained on A1AT coated surfaces were subtracted from the original values. Data are presented as relative fluorescent unit (RFU) measured at 525 nm with SD of two independent experiments. Differences with p < 0.05 were considered statistically significant and compared to no C3a treated samples (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01). Upper and lower asterisks indicate statistical significance of H1-AlexaFluor 488 binding in the presence of BSA or C3a (respectively) compared to binding of H1-AlexaFluor 488 alone.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Labeling

Journal: Brain Pathology

Article Title: Complement C3a induces axonal hypomyelination in the periventricular white matter through activation of WNT/β‐catenin signal pathway in septic neonatal rats experimentally induced by lipopolysaccharide

doi: 10.1111/bpa.12798

Figure Lengend Snippet: Primary antibodies used in experiments.

Article Snippet: To investigate whether C3a administration would activate WNT/β‐catenin pathway in the OPCs, XAV‐939 (a selective inhibitor against Wnt/β‐catenin‐mediated transcription by tankyrase1/2) (MedChemExpress, Cat. No. HY‐15147) was used to reverse the effect of C3a administration on the differentiation and maturation of OPCs.

Techniques: Concentration Assay

Journal: Brain Pathology

Article Title: Complement C3a induces axonal hypomyelination in the periventricular white matter through activation of WNT/β‐catenin signal pathway in septic neonatal rats experimentally induced by lipopolysaccharide

doi: 10.1111/bpa.12798

Figure Lengend Snippet: C3a inhibits the differentiation and maturation of OPCs in vitro. Immunofluorescence images of cultured OPCs showing the expression of MBP (A–D, green), NG2 (E–H, green) and DAPI (blue) at 4 days after the C3a, C3a + C3aRa and C3aRa treatment when compared with the corresponding control at the magnification of ×40 (n = 5). Panel I shows the immunoreactive bands of PLP (26 kDa), MBP (33 kDa), CNPase (47 kDa), MAG (69 kDa), NG2 (251 kDa), CC1 (310 kDa), Olig1 (26 kDa), Olig2 (37 kDa), SOX10 (49 kDa) and β‐actin (42 kDa) after C3a administration or C3a + C3aRa treatment or C3aRa treatment and the corresponding control (n = 6). Bar graphs (J–R) show the optical density of protein expression shown in I (n = 6). (S, T) Bar graphs show the percentage of MBP+/DAPI+ and NG2+/DAPI+ cells at 4 d after the C3a, C3a + C3aRa and C3aRa treatment when compared with the corresponding control (n = 5). For statistical analysis, one‐way ANOVA with Tukey's post hoc test was used for J–T and presented as the mean ± standard error of measurement (SEM). Scale bars: 20 μm. *P < 0.05, **P < 0.01.

Article Snippet: To investigate whether C3a administration would activate WNT/β‐catenin pathway in the OPCs, XAV‐939 (a selective inhibitor against Wnt/β‐catenin‐mediated transcription by tankyrase1/2) (MedChemExpress, Cat. No. HY‐15147) was used to reverse the effect of C3a administration on the differentiation and maturation of OPCs.

Techniques: In Vitro, Immunofluorescence, Cell Culture, Expressing, Control

Journal: Brain Pathology

Article Title: Complement C3a induces axonal hypomyelination in the periventricular white matter through activation of WNT/β‐catenin signal pathway in septic neonatal rats experimentally induced by lipopolysaccharide

doi: 10.1111/bpa.12798

Figure Lengend Snippet: C3a delays the differentiation and maturation of OPCs in vitro by activating the WNT/β‐catenin pathway. C3a administration activates the WNT/β‐catenin pathway in primary culture of OPCs. Panel A shows TCF4, β‐catenin, β‐catenin phosphorylation and AXIN2 immunoreactive bands after C3a treatment for 15 minutes, 30 minutes, 1, 2, and 4 h and the corresponding control (n = 6). Bar graphs (B–E) show the optical density of protein expression in A (n = 6). XAV939, an inhibitor of WNT/β‐catenin pathway, reverses the effect of C3a in primary culture of OPCs. Panel F shows the immunoreactive bands of PLP (26 kDa), MBP (33 kDa), CNPase (47 kDa), MAG (69 kDa), NG2 (251 kDa), CC1 (310 kDa), Olig1 (26 kDa), Olig2 (37 kDa), SOX10 (49 kDa) and β‐actin (42 kDa) after C3a, C3a + C3aRa, C3a + XAV939, XAV939 treatment and the corresponding control (n = 6). Bar graphs (G–O) show the optical density of protein expression in I (n = 6). For statistical analysis, one‐way ANOVA with Tukey's post hoc test was used for B–E and G–O and presented as the mean ± standard error of measurement (SEM). Scale bars: 20 μm. *P < 0.05, **P < 0.01.

Article Snippet: To investigate whether C3a administration would activate WNT/β‐catenin pathway in the OPCs, XAV‐939 (a selective inhibitor against Wnt/β‐catenin‐mediated transcription by tankyrase1/2) (MedChemExpress, Cat. No. HY‐15147) was used to reverse the effect of C3a administration on the differentiation and maturation of OPCs.

Techniques: In Vitro, Phospho-proteomics, Control, Expressing

Journal: Brain Pathology

Article Title: Complement C3a induces axonal hypomyelination in the periventricular white matter through activation of WNT/β‐catenin signal pathway in septic neonatal rats experimentally induced by lipopolysaccharide

doi: 10.1111/bpa.12798

Figure Lengend Snippet: Table of Contents Image (TOCI): A sketch map demonstrates the cellular and molecular mechanism associated with PWMD in the septic neonatal rats. Microglia and astrocyte are activated in the PWM after intraperitoneal injection of LPS and release massive amounts of C3a and IL‐1β. Then they will bind to their receptors (C3aR and IL‐1R1) on the OLs and may activate AKT signaling pathway. It will inhibit GSK3β, allowing β‐catenin cytoplasmic accumulation and binding to TCF4 in the nucleus for nuclear translocation to active WNT/β‐catenin signaling pathway. It can lead to the delay of maturation or differentiation of OPCs through inhibiting the differentiation transcription factor of OPCs. This would contribute to axonal hypomyelination in the PWM in the experimental induced septic neonatal rats. XAV‐939, a selective inhibitor of WNT/β‐catenin signaling pathway, which may indirectly stabilize Axin2 through Tankyrase1/2, can promote the maturation or differentiation of OPCs in vitro.

Article Snippet: To investigate whether C3a administration would activate WNT/β‐catenin pathway in the OPCs, XAV‐939 (a selective inhibitor against Wnt/β‐catenin‐mediated transcription by tankyrase1/2) (MedChemExpress, Cat. No. HY‐15147) was used to reverse the effect of C3a administration on the differentiation and maturation of OPCs.

Techniques: Injection, Binding Assay, Translocation Assay, In Vitro