Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 1 The expression of complement effectors C3a, C5a and C5b-9 in peripheral blood and renal biopsies. (A–C) Serum levels of complement C3a, C5a and C5b-9 were examined in healthy controls (n = 20), DM patients (n = 20) and DN patients (n = 20) by ELISA. (D) Representative IHC staining images of complement C3a, C5a and C5b-9 from healthy controls and DN patients. (E) Quantitative analysis of C3a, C5a and C5b-9 staining in renal tissues by Image-Pro Plus 6.0 (n = 10–15). (F) Serum levels of CRP in healthy control (n = 20), DM patients (n = 20) and DN patients (n = 20) detected by ELISA. 400× original magnification; scale bar, 100 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Control

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 2 Differences between human CRP and rat CRP in terms of structure and complement activation in vitro. (A) Human CRP and rat CRP migrated in 1/20 SDS-PAGE as pentamers and in SDS-PAGE as monomers. (B) The pentameric structure of human and rat CRP was shown by transmission electron microscopy (TEM). (C) Autologous complement activation of human CRP in human serum and rat CRP in rat serum (n = 6). (D) Interspecific complement activation of human CRP in rat serum and rat CRP in human serum (n = 6). Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Activation Assay, In Vitro, SDS Page, Transmission Assay, Electron Microscopy

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 3 The expression of complement C3a, C5a and C5b-9 in STZ-diabetic DKD rats. (A) Twenty-four-hour microalbuminuria was detected at 14-week with a commercial ELISA kit (n ≥ 8). (B) Blood urea nitrogen, serum CRE and urine CRE were monitored with an automated biochemistry analyzer (n ≥ 8). (C and D) Representative images and quantitative analysis of complement C3a, C5a and C5b-9 in rat kidney tissues by IHC staining (n = 4, more than 20 glomeruli). (E) Serum levels of complement C3a, C5a and C5b-9 were examined in rats with SZT-induced DKD by ELISA (n = 8). 400× original magnification; scale bar, 50 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 6 Anaphylatoxin C3a induced podocyte autophagy under high-glucose conditions. (A) Quantitative RT–PCR analysis of LC3B and p62 expression under low-glucose and high-glucose conditions with C3a and C5a treatment (n = 3). (B and C) Western blot and quantitative analysis of LC3B and p62 expression under high-glucose conditions with C3a treatment (n = 4). (D) Representative images of autophagy flux changes with C3a and C5a treatment after Ad-mCherry-GFP-LC3B infection (n = 3). 600× original magnification; scale bar, 10 µm. Data are presented as the means ± SEM, p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Infection

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 7 C3a-induced podocyte autophagy was regulated in stable CRP-overexpression and CRP-knockout cell lines. (A and B) Quantitative RT-PCR analysis of LC3B and p62 expression in the CRP-overexpression experiment and CRP-knockout experiment (n = 3). (C and D) Western blot and quantitative analysis of LC3B and p62 in the CRP-overexpression experiment and CRP-knockout experiment (n = 4). (E and F) Autophagic flux was detected in a stable CRP-overexpression cell line and a stable CRP-knockout cell line under high-glucose conditions with C3a treatment (n = 3). 600× original magnification; scale bar, 10 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Over Expression, Knock-Out, Quantitative RT-PCR, Expressing, Western Blot

Journal: The FASEB Journal

Article Title: C‐reactive protein inhibits C3a/C3aR‐dependent podocyte autophagy in favor of diabetic kidney disease

doi: 10.1096/fj.202200198r

Figure Lengend Snippet: FIGURE 8 CRP suppressed C3aR expression under high-glucose conditions. (A) western blot and quantitative analysis of C3aR expression in CRP-overexpression and CRP-knockout experiments (n = 4). (B) IF staining of LC3B in cultured CRP-overexpression and CRP-knockout cell lines with C3a treatment (n = 3). (C) A C3aR agonist active peptide was used to reverse changes in LC3B and p62 expression in CRP-FL cells (n = 4). (D) A C3aR antagonist SB290157 was used to reverse changes in LC3B and p62 expression in CRP- KO2 cells (n = 4). 200× original magnification; scale bar, 10 µm. Data are presented as the means ± SEM, and p < .05 was considered to be statistically significant

Article Snippet: Usually, the cells were seeded at 1 × 10 5/well in a 12- well plate, and then treated with low glucose (5 mM), high glucose (33 mM), recombinant mouse C3a (100 nM, HY- P7863, MCE, NJ, USA) and recombinant mouse C5a (100 nM, HY- P7695, MCE) for 24 h for qPCR and 72 h for Western blotting.

Techniques: Expressing, Western Blot, Over Expression, Knock-Out, Staining, Cell Culture

Journal: American Journal of Physiology - Renal Physiology

Article Title: Urine complement activation fragments are increased in patients with kidney injury after cardiac surgery

doi: 10.1152/ajprenal.00130.2019

Figure Lengend Snippet: The alternative pathway of complement is activated in mice after kidney ischemia-reperfusion (IR). C57BL/6 mice were subjected to 24 min of kidney ischemia or to sham surgery. A: kidneys were examined by immunofluorescence microscopy for C3b (green) and C4 (red), and nuclei were stained with DAPI (blue). C4 was seen in the glomeruli (arrowheads), and C3b was seen along the tubules (arrows) of sham-treated animals. C3b and C4 deposition was more intense in mice after ischemia and 24 h of reperfusion (T24 IR). C3 staining was still not seen in the glomeruli, however, and C4 staining was not seen in the tubulointerstitium. Control staining with isotype-matched antibodies was negative. Original magnification ×200. Scale bar = 50 μm. B: urine Ba fragments were measured by ELISA in urine samples collected before IR (pre) or after 3 h of reperfusion (3 h). Levels were significantly higher after IR. ***P < 0.001. C: Western blot analysis of urine for C4 showed that levels of intact C4 as well as C4 fragments were higher in samples collected after 3 h of reperfusion. WT, wild-type.

Article Snippet: The following primary antibodies were used in these studies: FITC-conjugated goat IgG to mouse complement C3 (MP Biomedicals, Santa Ana, CA), a monoclonal antibody (mAb) that only recognizes the iC3b and C3d activation fragments of C3 (mAb 3d29) ( 30 ), an antibody to C4 (Hycult Biotech, Uden, The Netherlands), goat anti-human properdin factor B (DiaSorin/Bio-Rad, Hercules, CA), and a biotinylated mAb to mouse factor B that binds to the Ba fragment ( 29 ).

Techniques: Immunofluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Translational Vision Science & Technology

Article Title: Systemic Sodium Iodate Injection as a Model for Expanding Geographic Atrophy

doi: 10.1167/tvst.14.1.9

Figure Lengend Snippet: List of Primary and Secondary Antibodies Used

Article Snippet: 1 , Goat anti-C3 , MP Biomedicals , 0855474 , 1:200.

Techniques:

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Schematic illustration of in vivo tumor immunotherapy enhanced by mRNA/HNPs through intravenous injection. H18 lipid, DOPE, cholesterol, DMG-PEG 2000 and mRNA were mixed to form mRNA/H 18 NPs with the special multilamellar concentric nanostructures. Following intravenous administration, mRNA/H 18 NPs demonstrated preferential adsorption of complement C3 proteins to form a characteristic protein corona, resulting in specific mRNA transfection in the spleen, especially in splenic dendritic cells. When encapsulating tumor antigen-encoding mRNA, the mRNA/H 18 NPs achieved precise transfection of the antigen mRNA in splenic dendritic cells. This targeted delivery stimulated dendritic cell maturation and subsequent antigen presentation, initiating robust T cell priming. The activated antigen-specific cytotoxic T lymphocytes then infiltrated into tumor tissues, ultimately inducing tumor cell elimination.

Article Snippet: Mouse Complement C3 was purchased from MedChemExpress.

Techniques: In Vivo, Injection, Adsorption, Transfection, Immunopeptidomics

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: In vivo splenic DC-specific transfection of mRNA/H 18 NPs and in vitro protein corona analysis of mRNA/H 18 NPs. (A) EGFP protein expression in splenic cell subsets of C57BL/6J mice 24 h post intravenous injection of different formulations. (B) The top 5 most abundant plasma proteins adsorbed on mRNA/H 18 NPs (C3: Complement C3; Ighm: Immunoglobulin heavy constant mu; Hbat1: Alpha-globin; Itih4: Inter alpha-trypsin inhibitor, heavy chain 4; Cnn2: Calponin). (C) Heatmap plot of major proteins in the protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. PBS group was used as a negative control. (D) Quantification of major adsorbed protein categories of different formulations. (E) Complement C3 abundance in protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. (F) Bioluminescence images of major organs and (G) Quantification of total bioluminescence flux in the spleen from C57BL/6J mice 6 h after intravenous injection of mLuc/H 18 NPs (mLuc dose of 0.25 mg kg −1 ). Mice were pre-treated with cobra venom factor (CVF) or PBS. (H) Fluorescence quantification of Cy5 mRNA delivered by uncoated or complement C3-coated Cy5-mRNA/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. (I) Bioluminescence intensity of luciferase protein translated from mRNA delivered by uncoated or complement C3-coated mLuc/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. Data were shown as mean ± SD (n = 3).

Article Snippet: Mouse Complement C3 was purchased from MedChemExpress.

Techniques: In Vivo, Transfection, In Vitro, Expressing, Injection, Clinical Proteomics, Negative Control, Combined Bisulfite Restriction Analysis Assay, Fluorescence, Incubation, Blocking Assay, Luciferase