Journal: Journal of Neuroinflammation

Article Title: Dual inhibition of IRAK1/TAK1 signaling in astrocytes reduces accelerated mortality in human APOE4 knock-in APPswe/PSEN1dE9/P301S-Tau triple transgenic mouse model

doi: 10.1186/s12974-025-03564-7

Figure Lengend Snippet: Inhibition of A1 astrocyte activation alleviated mitochondrial dysfunction in differentiating neurons. A Schematic overview of the in vitro study using differentiating MSP-1 newborn neurons. MSP-1 neurons were treated with cultured medium (CM) collected from MSP-1 astrocytes for 24 h. B Representative images of AT8 staining of MSP-1 neurons treated with TNF CM and TNF+Ans CM (scale bar, 10 μm). C Quantification of AT8 fluorescence intensity in each group, ( n = 3–5). D Representative electron microscopy images of MSP-1 neurons treated with Sham, TNF CM, TNF+Ans CM and TNF CM-C3aRA (scale bar, 2 μm (up), 1 μm (down)). N, nucleus, MT, normal mitochondria (blue), sMT, swollen mitochondria (yellow). E Quantification of mitochondrial length and the ratio of swollen mitochondria in each group (neuron number, 15–18; mitochondria number, 45–49). F Representative images of MT-1 staining of MSP-1 neurons treated with Sham, TNF CM, TNF+Ans CM and TNF CM-C3aRA (10 μM) (scale bar, 25 μm). G Quantification of MT-1 fluorescence intensity in each group ( n = 4–6). H Representative images of ROS staining of MSP-1 neurons treated with Sham, TNF CM, TNF+Ans CM and TNF CM-C3aRA (10 μM) (scale bar, 25 μm). I Quantification of ROS fluorescence intensity in each group ( n = 5–6). Data are presented as mean ± SEM ( C, G and I ) or presented as violin plots ( E ) (median: bold dashed line; quartiles (1/4 and 3/4: dashed lines)) and were analyzed by one-way ANOVA with Dunnett's multiple comparisons test ( C, G and I ) or Kruskal-Wallis test with Dunn's multiple comparisons test ( E ). All comparisons made were versus the TNF CM group. * p < 0.05, ** p < 0.01, *** p < 0.001. Some elements in panel A were created with BioRender.com

Article Snippet: For blocking experiments, necrostatin-1 (Nec-1) and the C3a receptor antagonist SB290157 (Selleck) were used.

Techniques: Inhibition, Activation Assay, In Vitro, Cell Culture, Staining, Fluorescence, Electron Microscopy

Journal: Journal of Fungi

Article Title: Vaginal Clinical Isolates of Candida albicans Differentially Modulate Complosome Activation in Vaginal Epithelial Cells

doi: 10.3390/jof11070501

Figure Lengend Snippet: Intracellular C3aR and C5aR1 analysis. A monolayer of VECs was infected or not (Mock) with the Colonizing strain (Col) or VVC strain (VVC) for 4 h. After incubation, intracellular C3aR ( a ) and C5aR1 ( b ) were analyzed by cytofluorimetric analysis. Data in the graphs show the mean ± SEM of C3aR- and C5aR1-positive cells (cell counts from 8000 live cells) obtained from 4 different experiments. ns—not significant; * p ≤ 0.05.

Article Snippet: The anti-C3aR antibody was obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Infection, Incubation

Journal: Journal of Fungi

Article Title: Vaginal Clinical Isolates of Candida albicans Differentially Modulate Complosome Activation in Vaginal Epithelial Cells

doi: 10.3390/jof11070501

Figure Lengend Snippet: Activation of complosome in VECs by C. albicans . Differential activation of C3, C3a and C3b ( a ) and C5 and C5a ( b ) and intracellular C3aR and C5aR1 involvement in VECs infected with the Colonizing or VVC-associated strain. Arrows indicate changes in the levels of complement factors and receptors after infection with either the Colonizing strain (left) or the VVC strain (right). Green indicates increased levels and red indicates lower levels as compared to resting levels. Created with BioRender (adapted from King B.C. and Blom A.M., 2024 ).

Article Snippet: The anti-C3aR antibody was obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Activation Assay, Infection

Journal: Aging Cell

Article Title: Age‐Related Complement C3 Drives Memory Impairments and Associated Neuropathologies in a Mouse Model

doi: 10.1111/acel.70145

Figure Lengend Snippet: Impairment of astrocyte insulin signaling and mitochondrial structure after activation of the C3a‐C3aR pathway. (a–c) Western blotting analysis of C3aR and CD11b expression in wild‐type (WT, 6 months old) and C3 +/+ mice (6 months old) ( n = 4 WT, n = 5 C3 +/+ ; unpaired Student's t ‐test). (d) ELISA analysis of C3a levels in brain tissue from WT ( n = 5) and C3 transgenic mice ( n = 5) (unpaired Student's t ‐test). (e, f) CCK‐8 assay of U251 cell survival after C3a (200 nM) and C3a + SB290157 treatments ( n = 6 per group; unpaired Student's t ‐test). (g) qPCR analysis of insulin receptor (IR) mRNA levels in U251 cells after C3a treatment ( n = 4 per group; unpaired Student's t ‐test). (h–j) Western blotting analysis of IR protein expression in U251 cells after C3a (200 nM) ( n = 7 per group ) and C3a + SB290157 treatments ( n = 5 per group; unpaired Student's t ‐test). (k–m) qPCR analysis of mitochondrial function‐related genes in U251 cells ( n = 4 per group; unpaired Student's t ‐test). (n) Quantitative analysis of mitochondrial area (unpaired Student's t ‐test). (o) Schematic representation of mitochondrial morphology as observed by transmission electron microscopy. (p, q) Measurement of reactive oxygen species (ROS) production in U251 cells: (p) C3a versus vehicle control; (q) C3a + C3aR antagonist (SB290157, 100 nM) versus vehicle control (unpaired Student's t ‐test). All data are presented as mean ± standard deviation (SD).

Article Snippet: Membranes were then immersed in a 5% bovine serum albumin solution (in Tris‐buffered saline plus 0.1% Tween‐20, pH 7.2) for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies against C3 (1:1000, ab200999, Abcam), C3aR (1:1000, bs‐2955R, Bioss, China), CD11b (1:1000, ab133357, Abcam), insulin receptor (1:2000, ABclonal, China), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 1:5000, MA5‐15738, Thermo Fisher Scientific), and tubulin (1:1000, T0023, Affinibody, China).

Techniques: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transgenic Assay, CCK-8 Assay, Transmission Assay, Electron Microscopy, Control, Standard Deviation