Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3aR ameliorates rotenone-induced neurodegeneration, synaptic loss, α-synuclein phosphorylation and cognitive deficits in mice. C57BL/6 mice were treated with rotenone for 3 weeks to establish PD models, with 30 min of SB290157 (C3aR inhibitor, 1 mg/kg, i.p.) administration after each rotenone injection. (A) Concentrations of C3a in the hippocampus of Con and Rot mice, measured by enzyme-linked immunosorbent assay (ELISA). n = 6. (B) Representative Western blot images and densitometric quantification of C3aR protein levels in the hippocampus and rotenone mice. n = 4. (C) The mRNA levels of C3aR in vehicle and rotenone mice. (D) Representative images of double-immunofluorescence staining with C3aR and Iba-1 or Neu-N antibodies and (E, F) the quantification of C3aR + Iba-1 + and C3aR + Neu-N + cells in vehicle and rotenone mice. n = 3. (G) The quantification of Neu-N + cell number and (H) optical density of PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. (I) The representative images of Neu-N and PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. n = 6. (J, K) Representative bands of Neu-N, PSD95 and ser129-phosphorylated α-synuclein and the quantification of blots in rotenone mice with or without C3aR inhibitor treatment. n = 4. (L) Representative images of ser129-phosphorylated α-synuclein and (M) the quantification of staining density in rotenone mice with or without C3aR inhibitor treatment. n = 3. (N–Q) Escape latency, traveled distance, first platform crossing latency and platform crossing number in rotenone mice with or without C3aR inhibitor treatment. n = 15; ∗ p < 0.05, ∗∗ p < 0.01 for comparison between Rot & Rot + C3aR inhibitor groups; Scale bar = 50 μm.

Article Snippet: The C3a ELISA kit was purchased from CUSABIO (CSB- E08511 m, Houston, TX, USA), and TUNEL assay kit was provided by KeyGEN (KGA7073-1, Nanjing, China.

Techniques: Inhibition, Phospho-proteomics, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Double Immunofluorescence Staining, Staining, Comparison

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) RT-PCR analysis of total versican in tubular cells treated with 20% patient serum (PS), 20% heat-inactivated patient serum (HIPS), 20% healthy serum (HS), or 20% heat-inactivated healthy serum (HIHS) for 48 hours (n = 3). (B) RT-PCR analysis of total versican in tubular cells treated with 20% PS, eculizumab, or SB290157 for 48 hours (n = 5). (C) Level of serum C3a in FSGS patients and controls (n = 20). (D) Level of urinary C3a in FSGS patients and controls (n = 20). (E) Immunofluorescence staining of C3a (red) and C3aR (green) in renal tissue of FSGS patients (n = 3). (F) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a or 20% PS for 48 hours (n = 3). Scale bars: 20 μm. For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for A, B, and F, and a 2-tailed Student’s t test was used for C and D. *P < 0.05 compared with control; #P < 0.05 compared with PS-, HS-, or C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) RT-PCR analysis of total versican in tubular cells treated with C3a and SB203580, PD098059, or MK2206 (n = 5). (B) RT-PCR analysis of versican V1, V0, and V3 in tubular cells treated with C3a and MK2206 (n = 3). (C) Western blot analysis of phospho-AKT (p-AKT) in tubular cells (n = 3). (D) Western blot analysis of nuclear β-catenin in tubular cells (n = 3). (E) Schematic of the β-catenin/TCF binding sites in the upstream sequence of the versican promoter and the constructed versican promoter–luciferase reporter plasmids. (F) ChIP analysis of the binding between β-catenin and the versican promoter in tubular cells treated with C3a (n = 3). (G) Normalized luciferase activity of reporter constructs in tubular cells cotransfected with N90-β-catenin plasmid (n = 3). (H) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells transfected with N90-β-catenin plasmid (n = 3). (I) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a and si-CTNNB1 (n = 3). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for A, B, and I, and a 2-tailed Student’s t test was used for G and H. *P < 0.05 compared with control; #P < 0.05 compared with C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Sequencing, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Level of serum creatinine in WT and C3aR-knockout (C3aR–/–) mice treated with Adriamycin (ADR) (n = 6). (B and C) Masson’s trichrome staining of renal sections in WT and C3aR–/– mice treated with ADR (n = 6). (D) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubulointerstitial tissues of WT and C3aR–/– mice treated with ADR (n = 6). (E and F) Immunohistochemical staining of versican in renal tissues of WT and C3aR–/– mice treated with ADR (n = 6). (G) Level of urinary C3a in WT and C3aR–/– mice treated with ADR (n = 6). (H) Immunofluorescence staining of C3a in WT and C3aR–/– mice treated with ADR (n = 6). (I) Western blot analysis of p-AKT in tubulointerstitial tissues of WT and C3aR–/– mice treated with ADR (n = 6). (J) Western blot analysis of nuclear β-catenin (n = 6). (K) ChIP analysis of the binding between β-catenin and the versican promoter in tubulointerstitial tissues (n = 6). (L) Western blot analysis of versican V1 in tubulointerstitial tissues (n = 6). Scale bars: 20 μm (E and H). Scale bars: 30 μm (B). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for A, C, D, F, and G. *P < 0.05 compared with control WT mice; #P < 0.05 compared with ADR-treated WT mice.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Knock-Out, Staining, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Immunofluorescence, Western Blot, Binding Assay, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Level of serum suPAR in FSGS patients (n = 20). (B) Level of urinary suPAR in FSGS patients (n = 20). (C) Immunohistochemical staining of uPAR in renal tissues of FSGS patients (n = 5). (D) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and uPAR-blocking antibody (n = 5). (E) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with C3a and suPAR (n = 5). (F) RT-PCR analysis of ITGB6 in tubulointerstitial tissues of FSGS patients (n = 20). (G) IP analysis of the binding between suPAR and ITGB6 in tubular cells treated with 20% PS (n = 3). (H) Immunofluorescence staining of uPAR (green) and ITGB6 (red) in tubular cells treated with 20% PS (n = 3). Scale bars: 20 μm (C and H). (I) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and si-ITGB6 (n = 5). (J) Rac1 activation assay in tubular cells treated with 20% PS and si-ITGB6 (n = 3). (K) RT-PCR analysis of total versican, versican V1, V0, and V3 in tubular cells treated with 20% PS and si-Rac1 (n = 5). For statistical analysis, a 2-tailed Student’s t test was used for A, B, and F, and 1-way ANOVA with Tukey’s post hoc test was used for D, E, I, J, and K. *P < 0.05 compared with control; #P < 0.05 compared with PS- or C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Binding Assay, Immunofluorescence, Activation Assay, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Splicing factors predicted to bind to the 5′ end of versican exon 7 with the SpliceAid 2 database. (B) IP analysis of the binding between Rac1 and SRp40 in tubular cells treated with 20% PS (n = 3). (C) Immunofluorescence staining of SRp40 (green), Rac1 (red), and DAPI (blue) in tubular cells treated with 20% PS (n = 3). Scale bars: 20 μm. (D) RIP analysis of the binding of SRp40 and Rac1 to the 5′ end of versican exon 7 in tubular cells treated with 20% PS (n = 5). (E) RIP analysis of the binding of Rac1 to the 5′ end of versican exon 7 in tubular cells treated with 20% PS and si-SRp40 (n = 3). (F) RIP analysis of the binding of SRp40 to the 5′ end of versican exon 7 in tubular cells treated with 20% PS and si-Rac1 (n = 3). (G) Rac1 activity in tubular cells treated with 20% PS and si-SRp40 (n = 3). (H) RIP analysis of the binding of U2AF1 to the 3′ splice site of versican intron 6 in tubular cells treated with 20% PS and pGEMT-SRp40 plasmid or suPAR-blocking antibody (n = 5). (I) PCR analysis of versican V0 and V1 in tubular cells treated with 20% PS and pGEMT-SRp40 plasmid or suPAR-blocking antibody (n = 3). (J) RIP analysis of the binding of U2AF1 to the 3′ splice site of versican intron 6 in tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 5). (K) PCR analysis of versican V0 and V1 in tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 3). For statistical analysis, 1-way ANOVA with Tukey’s post hoc test was used for D–F, G, H, and J. *P < 0.05 compared with control; #P < 0.05 compared with PS- or C3a-treated cells.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Binding Assay, Immunofluorescence, Staining, Activity Assay, Plasmid Preparation, Blocking Assay, Control

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: (A) Model of the 34-nt complementary sequence in intron 6 (site 1) and intron 8 (site 2) of versican pre-mRNA. (B) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells (n = 3). (C) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells treated with LNA antisense oligonucleotides targeting either site 1 or site 2 (n = 3). (D) PCR analysis of versican V3 in tubular cells (n = 3). (E) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells transfected with Rac1Q61L plasmid and si-SRp40 (n = 3). (F) PCR analysis of versican V3 in tubular cells transfected with Rac1Q61L plasmid and si-SRp40 (n = 3). (G) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells treated with 20% PS and si-Rac1 or suPAR-blocking antibody (n = 3). (H) PCR analysis of versican V3 in tubular cells treated with 20% PS and si-Rac1 or suPAR-blocking antibody (n = 3). (I) RAP analysis of the intron 6/8 interaction in versican pre-mRNA of tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 3). (J) PCR analysis of versican V3 in tubular cells treated with C3a and Rac1Q61L plasmid or suPAR (n = 3).

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Sequencing, Transfection, Plasmid Preparation, Blocking Assay

Journal: JCI Insight

Article Title: C3a and suPAR drive versican V1 expression in tubular cells of focal segmental glomerulosclerosis

doi: 10.1172/jci.insight.122912

Figure Lengend Snippet: C3a promotes the transcription of versican by activating the AKT/β-catenin pathway in tubular cells. suPAR binds to ITGFB6 and activates Rac1, which translocates into the nucleus and binds to SRp40 at the 5′ end of exon 7 of versican pre-mRNA. This binding not only inhibits the 3′-end splicing of intron 6 but also inhibits the base-pair interaction between intron 6 and intron 8, which leads to the formation of versican V1.

Article Snippet: A sandwich ELISA was performed according to the manufacturer’s protocol to quantify human C3a (KA1020, Novus Biologicals), mouse C3a (NBP2-70037, Novus Biologicals), human uPAR (DY807, R&D Systems), and mouse uPAR (DY531, R&D Systems).

Techniques: Binding Assay