c5 in 1  (MedChemExpress)

Journal: Biomolecules

Article Title: Selective Complement Inhibition in Anti-p200 Pemphigoid: Immune Infiltrate Profiles and Therapeutic Implications Compared to Bullous Pemphigoid

doi: 10.3390/biom16020182

Figure Lengend Snippet: Inhibition of complement fixation by selective inhibitors in an in vitro model of anti-p200 pemphigoid. Human skin cryosections were incubated with heat-inactivated, pooled sera from three anti-p200 pemphigoid patients exhibiting strong C3c/C5-fixating activity, followed by a complement source. Increasing concentrations of selective inhibitors targeting C1s (sutimlimab), C3 (compstatin), C5 (tesidolumab), or C5aR1 (avacopan) were added to the complement source. EDTA and tinzaparin sodium served as positive controls for complement inhibition. BMZ complement fixation (C3c or C5; green) was assessed by IF microscopy and semi-quantified relative to untreated controls (no inhibitor). Sutimlimab and compstatin dose-dependently suppressed C3c deposition, while tesidolumab and avacopan led to almost complete inhibition of C5 complement fixation at all tested concentrations. In contrast, no complement fixation was observed in the presence of normal human serum (NHS). Nuclei were visualized by DAPI (blue) counterstaining. Semi-quantitative analyses were performed using three independent experiments. **** p < 0.0001; ** p < 0.01; * p < 0.05. Scale bar, 100 μm.

Article Snippet: Sections were then stained with FITC-conjugated rabbit anti-human C3c (Dako, Carpinteria, CA, USA) or FITC-conjugated human complement C5 AssayLite ® (AssayPro LLC, St. Charles, MO, USA) antibodies for 1 h at RT.

Techniques: Inhibition, In Vitro, Incubation, Activity Assay, Microscopy

Journal: Biomolecules

Article Title: Selective Complement Inhibition in Anti-p200 Pemphigoid: Immune Infiltrate Profiles and Therapeutic Implications Compared to Bullous Pemphigoid

doi: 10.3390/biom16020182

Figure Lengend Snippet: Targeted inhibition of complement deposition induced by BP autoantibodies in an in vitro model. Human skin cryosections were incubated with pooled, heat-inactivated sera from three BP patients with strong C3c/C5 fixation (green). Selective complement inhibitors, including anti-C1s antibody (sutimlimab), C3 inhibitor (compstatin), anti-C5 antibody (tesidolumab), or C5aR1 inhibitor (avacopan), as well as EDTA and tinzaparin sodium (positive controls for complete complement inhibition) were added alongside hirudin plasma as a complement source. Tissues were stained for C3c or C5 deposition, and percentage inhibition of complement deposition was semi-quantified relative to untreated samples. A dose-dependent inhibition of C3c deposition was observed with compstatin, while all other compounds led to a nearly complete C3c or C5 inhibition along the BMZ at all tested concentrations. Normal human serum (NHS) did not induce complement fixation. Nuclei were visualized by DAPI (blue) counterstaining. Data from three independent experiments were used for semi-quantitative analysis. **** p < 0.0001; * p < 0.05. Scale bar: 100 μm.

Article Snippet: Sections were then stained with FITC-conjugated rabbit anti-human C3c (Dako, Carpinteria, CA, USA) or FITC-conjugated human complement C5 AssayLite ® (AssayPro LLC, St. Charles, MO, USA) antibodies for 1 h at RT.

Techniques: Inhibition, In Vitro, Incubation, Clinical Proteomics, Staining

Journal: Nature Communications

Article Title: Potent neutralization by antibodies targeting the MPXV A28 protein

doi: 10.1038/s41467-025-66344-0

Figure Lengend Snippet: a Neutralization kinetics of VACV IHDJ by anti-MPXV mAbs at IC 50 concentrations with 1.5% NHS (3% during pre-incubation). NHS and mAbs were added at 10-min intervals from 2 h before to 2 h after infection. b Neutralization in the presence of 1.5% NHS (3% during pre-incubation) and 6 consecutive 4-fold dilutions of C5-IN-1 starting from 10 μM. c Same as ( b ) but with 8 consecutive 5-fold dilutions of a C3 inhibitor AMY-101 starting from 10 μM. d Neutralization in the presence of 1.5% NHS (3% during pre-incubation) or C1q- or C3-depleted human serum. e C1q deposition on VACV IHDJ virions ( ~ 5 × 10⁶ PFU) incubated with 1.5% NHS and 10 μg/mL mAbs. Left panel: flow cytometry plots, pre-gated for virions using GFP followed by staining for C1q-AF647. Right panel: median fluorescence intensity. f-h TEM visualization of mAb and complement factors C1q, and C3 deposition on virions. Purified MV particles were incubated with 10 μg/mL of either mAb 7M1162 anti-H3 ( f ), 8M2110 anti-A28 ( g ) or MGO53 isotype control ( h ) and 1.5% NHS. IgG, C1q, and C3 deposition were visualized using anti-human IgG gold nanoparticles (10 nm) or secondary mouse anti-C1q and mouse anti-C3, followed by labeling with anti-mouse gold nanoparticles (20 nm). Anti-H3 and anti-A28 mAbs are shown in magenta and green, respectively; anti-M1 (7D11) in black; MGO53 in gray. IC 50 concentrations for panels ( a – d ) were: 15 μg/mL (7M1162), 0.125 μg/mL (8M2110), 1.25 μg/mL (10M2146), and 62.5 μg/mL (10M2154). Spearman correlation was used in ( a ) to determine the relationship between pre-incubation times and neutralization. Standard curves were determined by fitting values using simple linear regression for ( a ) and Inhibitor vs. normalized response (Variable slopes) nonlinear regression for ( b - c ). Standard deviation of the mean is shown for all values in ( a – d ). The percentages of complement sources indicated represent the final concentrations in the infected wells. Data are representative of two independent experiments with similar results using technical replicates: n = 3 for ( a ), n = 5 for ( b ), n = 6 for ( c ), and n = 3 for ( d ). Source data are provided as a Source Data file.

Article Snippet: For experiments involving selective complement inhibitors, mAbs and NHS mixtures were incubated with either 6 consecutive 4-fold dilutions, starting from 10 μM of a selective complement C5 inhibitor, C5-IN-1 (Compound 7- MedChemExpress), or 8 consecutive 5-fold dilutions, starting from 10 μM of a selective complement C3 inhibitor, CP40 (AMY-101- MedChemExpress).

Techniques: Neutralization, Incubation, Infection, Flow Cytometry, Staining, Fluorescence, Purification, Control, Labeling, Standard Deviation

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Effects of blocking the C5a/C5aR1 signaling on the early stage of lung fibrosis: ( A ) Scheme for the BLM-induced lung fibrosis. The mice were treated with PMX53 (1 mg/kg) or vehicle 1 h before BLM exposure on day 0, and then once a day. Mice were euthanized on day 7 after BLM modeling. ( B ) Weight changes of the mice; n = 8 for each group. ( C ) Lung coefficients of the mice; n = 8 for each group. ( D ) H&E staining of the lung tissues. Representative images are shown. Original magnification: 200×. ( E ) The number of nuclei per high-power field of lung tissues; n = 8 for each group. ( F ) The inflammatory index of the alveoli; n = 8 for each group. ( G ) The Ashcroft score; n = 8 for each group. ( H , I ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 in lung tissues was quantified by qPCR; n = 6 for each group. ( J ) The expression of αSMA protein in lung tissues. Representative immunohistochemical plots and statistical plots are shown. Original magnification: 200×; n = 8 for each group. ( K , L ) Immunohistochemical staining of collagen I in lung tissues. Representative images and relative IOD analyses are shown. Original magnification: 200×; n = 8 for each group. ( M ) Masson staining of the lung tissues. Representative images and collagen volume fraction analyses are shown. Original magnification: 200×; n = 8 for each group. ( N , O ) WB analysis of the ACSL4 and αSMA protein in lung tissues. Representative bands and statistical plots are shown; n = 6 for each group. ( P ) The expression of Acsl4 in lung tissues was quantified by qPCR; n = 6 for each group. ( Q ) Immunohistochemical staining of ACSL4 protein in the lung tissues. Representative images and relative IOD analyses are shown; n = 8 for each group. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA followed by adjustments for multiple comparisons in panel ( B ), or Student’s t -test in panels ( C , E – J , L , M , O – Q ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Blocking Assay, Staining, Expressing, Immunohistochemical staining

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Inhibition of C5a/C5aR1 signaling reduced ACSL4 levels and attenuated lung inflammation and fibrosis in the chronic stage of pulmonary fibrosis: ( A ) Scheme of BLM-induced lung fibrosis. Mice were euthanized on day 21 after BLM modeling. ( B ) Weight changes of the mice; n = 6 for each group. ( C ) Lung coefficients of the mice; n = 6 for each group. ( D ) H&E staining of the lung tissues. Representative images are shown. Original magnification: 200×. ( E ) Number of nuclei per high-power field of the lung tissues; n = 6 for each group. ( F ) The inflammatory index of the alveoli; n = 6 for each group. ( G ) The Ashcroft score; n = 6 for each group. ( H , I ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 in lung tissues was quantified by qPCR; n = 5 for each group. ( J ) Immunohistochemical staining of αSMA in lung tissues. Representative images and relative IOD analyses are shown. Original magnification: 200×; n = 6 for each group. ( K , L ) Immunohistochemical staining of collagen I in lung tissues. Representative images and relative IOD analyses are shown. Original magnification: 200×; n = 6 for each group. ( M ) Masson staining of lung tissues. Representative images and collagen volume fraction analyses are shown. Original magnification: 200×; n = 6 for each group. ( N , O ) WB analysis of the ACSL4 and αSMA proteins in lung tissues. Representative bands and statistical plots are shown; n = 6 for each group. ( P ) The expression of Acsl4 in lung tissues was quantified by qPCR; n = 6 for each group. ( Q ) Immunohistochemical staining of ACSL4 in lung tissues. Representative images and relative IOD analyses are shown; n = 6 for each group. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA followed by adjustments for multiple comparisons in panel ( B ), or Student’s t -test in panels ( C , E – J , L , M , O – Q ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Inhibition, Staining, Expressing, Immunohistochemical staining

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Blocking ACSL4 reduced C5a/C5aR1 signaling-induced activation and migration of lung fibroblasts: ( A ) The expression of Acsl4 was quantified by qPCR; n = 6 for each group. ( B ) WB analysis of ACSL4 in lung fibroblasts. Representative bands and statistical plots are shown; n = 6 for each group. ( C , D ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 was quantified by qPCR; n = 6 for each group. ( E ) WB analysis of αSMA in lung fibroblasts. Representative bands and statistical plots are shown; n = 6 for each group. ( F ) The expression of αSMA was tested by immunofluorescence. Representative plots are shown. Original magnification: 400×; αSMA (green) and DAPI (blue). ( G ) The cell scratch assay was used to assess lung fibroblasts’ migration. Representative images are shown. ( H ) The transwell assay. Representative plot and statistical plots of the transwell assay results are shown; n = 3 for each group. ( I ) The proliferation of fibroblasts was tested by CCK8. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t -test in panels ( A , B ), one-way ANOVA followed by adjustments for multiple comparisons in panels ( C – E , H ), or two-way ANOVA followed by adjustments for multiple comparisons in panel ( I ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Blocking Assay, Activation Assay, Migration, Expressing, Immunofluorescence, Wound Healing Assay, Transwell Assay

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Blockade of calcium signaling attenuated ACSL4 expression induced by the C5a/C5aR1 signaling: ( A ) The calcium concentration of lung fibroblasts was tested by calcium imaging. Representative plots and statistical plots are shown; n = 4 for each group. ( B , C ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 was quantified by qPCR; n = 6 for each group. ( D ) WB analysis of ACSL4 and αSMA in lung fibroblasts. Representative bands and statistical plots are shown; n = 6 for each group. ( E ) The expression of αSMA was tested by immunofluorescence. Plots are shown. Original magnification: 400×; αSMA (green) and DAPI (blue). ( F , G ) The horizontal migration ability of lung fibroblasts was tested by the cell scratch assay. Representative plots and statistical plots are shown; n = 4 for each group. ( H ) The spatial migration ability of lung fibroblasts was tested by transwell assay. Representative plot and statistical plot are shown; n = 5 for each group. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, , **** p < 0.0001 by one-way ANOVA followed by adjustments for multiple comparisons in panels ( A – C , H ), Student’s t -test in panel ( D ), or two-way ANOVA followed by adjustments for multiple comparisons in panel ( G ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Expressing, Concentration Assay, Imaging, Immunofluorescence, Migration, Wound Healing Assay, Transwell Assay