Journal: BMC Cardiovascular Disorders

Article Title: Complement C3a/C3aR inhibition alleviates the formation of aortic aneurysm in Marfan syndrome mice

doi: 10.1186/s12872-024-04077-6

Figure Lengend Snippet: Expression of C3aR was upregulated in the aorta of MFS mice. (A) the mRNA levels of C3aR in 3-month-old WT and FBN1 C1041G/+ aortas were accessed by qRT-PCR ( n = 4 per group). (B , C) IHC staining for C3aR in the aortas of 3-month-old WT and FBN1 C1041G/+ mice ( n = 6 per group). ** P < 0.01, vs. the WT group. (D) the mRNA levels of Il1b , Ccl2 , Ccl5 , Spp1 in PBS, recombinant C3a (1ug/mL) stimulated macrophages were accessed by qRT-PCR. ( n = 4 per group). (E , F) CBA assay was used to detect the concentration of IL-1β (E) and CCL2 (F) in culture medium of PBS or recombinant C3a stimulated macrophages. ( n = 4 per group). The data were analyzed using the nonparametric Kruskal-Wallis H test. * P < 0.05, ** P < 0.01, vs. PBS group

Article Snippet: For IHC staining, the sections were incubated with 3% H 2 O 2 for 10 min at room temperature and then blocked with serum for 30 min. After blocking, the sections were incubated with primary antibodies [anti-C3aR antibody (1:400 dilution; Santa Cruz Biotechnology), anti-Mac2 antibody (1:400 dilution; Santa Cruz Biotechnology), or anti-IL-1β antibody (1:200 dilution; Santa Cruz Biotechnology)] at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Recombinant, Concentration Assay

Journal: BMC Cardiovascular Disorders

Article Title: Complement C3a/C3aR inhibition alleviates the formation of aortic aneurysm in Marfan syndrome mice

doi: 10.1186/s12872-024-04077-6

Figure Lengend Snippet: C3aR antagonist (C3aRA) reduces aortic dilatation and elastic fiber rupture of MFS mice. (A) (B-C) Representative images of ultrasound images of the ascending aortas ( B ) and abdominal aortas ( C ) of WT, MFS controls and C3aRA treated MFS mice. (D-E) The diameters of ascending aortas (AsAo) and abdominal aortas (AbAo) ( n = 6 per group). (F) Representative images of EVG elastic fiber staining of ascending aortic section from WT, MFS controls and C3aRA treated MFS mice. Scale bar, 50 μm. (G-H) Quantification of aortic medial thickness (G) and elastic fiber breaks (H) in the ascending aortic sections ( n = 6 per group). The data were analyzed using the nonparametric Kruskal-Wallis H test. ** P < 0.01 vs. WT group; ++ P < 0.01 vs. MFS PBS group

Article Snippet: For IHC staining, the sections were incubated with 3% H 2 O 2 for 10 min at room temperature and then blocked with serum for 30 min. After blocking, the sections were incubated with primary antibodies [anti-C3aR antibody (1:400 dilution; Santa Cruz Biotechnology), anti-Mac2 antibody (1:400 dilution; Santa Cruz Biotechnology), or anti-IL-1β antibody (1:200 dilution; Santa Cruz Biotechnology)] at 4 °C overnight.

Techniques: Staining

Journal: BMC Cardiovascular Disorders

Article Title: Complement C3a/C3aR inhibition alleviates the formation of aortic aneurysm in Marfan syndrome mice

doi: 10.1186/s12872-024-04077-6

Figure Lengend Snippet: Schematic illustration of the C3a-C3aR pathway contributing to the formation of aortic aneurysm in MFS mice

Article Snippet: For IHC staining, the sections were incubated with 3% H 2 O 2 for 10 min at room temperature and then blocked with serum for 30 min. After blocking, the sections were incubated with primary antibodies [anti-C3aR antibody (1:400 dilution; Santa Cruz Biotechnology), anti-Mac2 antibody (1:400 dilution; Santa Cruz Biotechnology), or anti-IL-1β antibody (1:200 dilution; Santa Cruz Biotechnology)] at 4 °C overnight.

Techniques:

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: The hub genes detection form NRGs and DEGs from Target-OS. ( A ) The Venn diagram illustrates the presence of 24 differentially expressed genes (DEGs) resulting from the overlap between 538 neutrophil-related genes (NRGs) and 161 up-regulated DEG mRNAs from Target-OS. ( B ) The most significant candidate genes were identified through the implementation of univariate Cox regression analyses. ( C ) Prognostic signature genes were selected based on their association with survival outcomes, as demonstrated by Kaplan–Meier (KM) survival curves in Target-OS. ( D , E ) The LASSO algorithm was employed to select hub genes. ( F ) The LASSO algorithm identified two genes, namely C3AR1 and FCER1G, as significant.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques:

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: ESTIMATE evaluation based on the two hub genes. ( A – H ) In both the TARGET-OS and GSE21257 cohorts, a comparison was made between high and low samples regarding the stromal scores, immune scores, ESTIMATE scores, and tumor purity. Additionally, it was observed that C3AR1 or FCER1G exhibited a significant positive correlation with stromal scores, ESTIMATE scores, and immune scores, while displaying a strong negative correlation with tumor purity in both cohorts. ** p < 0.01, ****: p < 0.0001.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques: Comparison

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: Evaluation of immunomodulators based on the two hub genes. ( A , B ) Heatmap displaying enrichment of immunomodulators through 7 algorithms between high- and low groups according to C3AR1 and FCER1G levels in the TARGET-OS ( A ) and GSE21257 cohorts ( B ). ns: p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques:

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: FCER1G/C3AR1 correlated KEGG NSP in OS. ( A ) The correlation between FCER1G and KEGG NSP genes was examined in the TARGET-OS dataset. ( B ) The correlation between FCER1G and KEGG NSP scores was investigated in the TARGET-OS dataset. ( C ) A comparison was made between the KEGG NSP scores in subgroups with high and low FCER1G expression in the TARGET-OS dataset. ( D ) The expression of FCER1G was compared between subgroups with high and low KEGG NSP scores in the TARGET-OS dataset. ( E ) The correlation between FCER1G and KEGG NSP genes was examined in the GSE21257 dataset. ( F ) The correlation between FCER1G and KEGG NSP scores was investigated in the GSE21257 dataset. ( G ) A comparison was made between the KEGG NSP scores in subgroups with high and low FCER1G expression in the GSE21257 dataset. ( H ) The expression of FCER1G was compared between subgroups with high and low KEGG NSP scores in the GSE21257 dataset. ( I ) The correlation between C3AR1 and KEGG NSP genes was examined in the TARGET-OS dataset. ( J ) The correlation between C3AR1 and KEGG NSP scores was investigated in the TARGET-OS dataset. ( K ) A comparison was made between the KEGG NSP scores in subgroups with high and low C3AR1 expression in the TARGET-OS dataset. ( L ) The expression of C3AR1 was compared between subgroups with high and low KEGG NSP scores in the TARGET-OS dataset. ( M ) The correlation between C3AR1 and KEGG NSP genes was examined in the GSE21257 dataset. ( N ) The correlation between C3AR1 and KEGG NSP scores was investigated in the GSE21257 dataset. ( O ) A comparison was made between the KEGG NSP scores in subgroups with high and low C3AR1 expression in the GSE21257 dataset. ( P ) The expression of C3AR1 was compared between subgroups with high and low KEGG NSP scores in the GSE21257 dataset. ****: p < 0.0001.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques: Comparison, Expressing

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: SPI1 was identified as a potential key TF in OS. ( A ) The heatmap illustrates the identification of 18 transcription factors (TFs) using the DoRothEA R package in scRNA-seq data. ( B ) The volcano plot generated from the analysis of scRNA-seq data reveals the presence of eight differentially expressed genes (DEGs) that exhibit significant differences between aneuploid and diploid groups. ( C , D ) The identification of potential transcription factors (TFs) that may regulate the C3AR1 and FCER1G genes involves screening two databases (JASPAR and ChEA) through the utilization of NetworkAnalyst 3.0. ( E – H ) The expression levels of SPI1 and RUNX1 were found to be decreased in both the TARGET-OS and GSE21257 datasets. ( I – L ) The Kaplan–Meier survival plots demonstrate the overall survival (OS) of the SPI1 and RUNX1 genes in both the TARGET-OS and GSE21257 datasets.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques: Generated, Expressing

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: Evaluation of C3AR1, FCER1G and SPI1 in OS animal model. ( A – C ) The three genes (C3AR1 ( A ), FCER1G ( B ) and SPI1 ( C )) showed significant upregulation in OS mice tissue samples compared to normal tissue samples. ( D , E ) Correlation circle pipes and light image show the correlations among C3AR1, FCER1G and SPI1 genes in OS mice tissues. ( F ) The potential transcription factor regulatory network in OS was investigated. Data were represented as mean with SD.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques: Animal Model

Journal: Biomedicines

Article Title: Exploring the Role of Neutrophil-Related Genes in Osteosarcoma via an Integrative Analysis of Single-Cell and Bulk Transcriptome

doi: 10.3390/biomedicines12071513

Figure Lengend Snippet: IHC for C3AR1, FCER1G and SPI1. ( A – C ) IHC for C3AR1, FCER1G, and SPI1 in the normal tissue samples (20× magnification). ( D – F ) IHC for C3AR1, FCER1G and SPI1 in the OS mice tissues (20× magnification). ( G – I ) The violin plots exhibit higher levels of staining for C3AR1, FCER1G and SPI1 protein expression in OS tissues as compared to normal tissue samples. AOD = Average optical density. * p < 0.05.

Article Snippet: Whereafter, anti-C3AR1 (bs-2955R 1:200; Bioss), anti-FCER1G (bs13167R 1:200; Bioss), and anti-SPI1 (66618-2-lg 1:1000; Proteintech) were incubated at 4 °C overnight using antibody diluent solution (Life-iLab, Shanghai, China).

Techniques: Staining, Expressing

Journal: Clinical and Experimental Medicine

Article Title: Complement C3a/C3aR and C5a/C5aR deposits accelerate the progression of advanced IgA nephropathy to end-stage renal disease

doi: 10.1007/s10238-024-01410-3

Figure Lengend Snippet: Immunohistochemical staining suggests increased expression of C3a, C3aR, C5a, C5aR in advanced IgAN (× 400), with significant differences between ESRD and non-ESRD groups. AOD, average optical density. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The antibodies used in this study included anti-C3a antibody (Abcam, ab36385, 1:200), anti-C3aR antibody (Abcam, ab140788, 1:200), anti-C5a antibody (Abcam, ab281923, 1:200), and anti-C5aR antibody (Abcam, ab252435, 1:1000).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Clinical and Experimental Medicine

Article Title: Complement C3a/C3aR and C5a/C5aR deposits accelerate the progression of advanced IgA nephropathy to end-stage renal disease

doi: 10.1007/s10238-024-01410-3

Figure Lengend Snippet: Spearman correlation analysis between C3a/C3aR, C5a/C5aR and baseline eGFR, baseline 24 h-UP, ΔeGFR/M. The results suggested that C3a ( A , r = − 0.58, P < 0.001), C3aR ( D , r = − 0.36, P = 0.002), C5a ( G , r = − 0.59, P < 0.001) and C5aR ( J , r = − 0.35, P = 0.003) were all negatively correlated with baseline eGFR, while positively with baseline 24 h-UP ( B , E , H , K ). Deposits of C3a/C3aR and C5a/C5aR could accelerate the mean rate of renal function decline ( C , F , I , L )

Article Snippet: The antibodies used in this study included anti-C3a antibody (Abcam, ab36385, 1:200), anti-C3aR antibody (Abcam, ab140788, 1:200), anti-C5a antibody (Abcam, ab281923, 1:200), and anti-C5aR antibody (Abcam, ab252435, 1:1000).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Mingjing granule inhibits the subretinal fibrovascular membrane of two-stage laser-induced neovascular age-related macular degeneration in rats

doi: 10.3389/fphar.2024.1384418

Figure Lengend Snippet: Effect of MG on the complement system. RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelial layer; * model vs. normal; # anti-VEGF/MG + anti-VEGF vs. model; and &MG + anti-VEGF vs. anti-VEGF; data are represented as mean ± SD; immunofluorescence: n = 5; qRT-PCR: n = 3. (A) Image of immunofluorescence staining, rows represent different factors, and columns represent different groups. (B) Immunofluorescence expression levels of C3aR: * p = 0.000, # p = 0.000 (anti-VEGF) and p = 0.000 (MG + anti-VEGF), & p = 0.015; expression levels of (C) C3aR mRNA: * p = 0.004, # p = 0.014 (MG + anti-VEGF); and (D) C3a mRNA: * p = 0.000, # p = 0.002 (anti-VEGF) and p = 0.000 (MG + anti-VEGF), & p = 0.000.

Article Snippet: Rabbit anti-transforming growth factor-beta 1 (TGF-β1) antibody (No: 21898) was purchased from Wuhan Sanying Biotechnology Co., Ltd., China; rabbit anti-C3aR antibody (No: bs2955R) was purchased from Beijing Boosen Biological Co., Ltd., China; DAPI antibody (No: AR1176) was purchased from Wuhan Borges De Biological Engineering Co., Ltd., China; RNeasy plus Universal Mini kits (No: 73404) were purchased from QIAGEN, Germany. iScript cDNA Synthesis Kit (No: 1708891) and iTaq Universal SYBR Green Supermix (No: 1725121) were purchased from Bio-Rad, United States.

Techniques: Immunofluorescence, Quantitative RT-PCR, Staining, Expressing

Journal: Journal of Neuroinflammation

Article Title: Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging

doi: 10.1186/s12974-024-03130-7

Figure Lengend Snippet: Altered expression of complement pathway components by old hippocampal microglia, especially in females. ( a ) Complement System pathway identified by performing IPA analysis of old versus young female, old versus young male, and old female versus old male microglia. Dashed line: -log 10 (p-value) cutoff of 1.3 ( p < 0.05). ( b ) Expression of the complement pathway genes C3 , C3ar1 , Cd55 and Ctsl . Data are presented as mean (SD) FPKM values. n = 5/group. ( c ) Complement gene expression by homeostatic (young and old) microglia and old DAM . Dot size shows the percentage of cells expressing the genes, and the color intensity scale indicates average gene expression by all cells in the cluster. ( d , e ) Complement gene expression by WT and 5XFAD homeostatic microglia and 5XFAD DAM from male and female mice (1 male and 2 females; d ), and homeostatic microglia and DAM1 and DAM2 subsets (1 male and 2 females combined; e ) . ( f , g ) C3aR protein expression in the hippocampus of young and old, male and female mice. Representative immunoblot images ( f ; arrow: C3aR band) and quantification of C3aR expression ( g ) are shown. n = 5/group. Data are presented as mean (SD). ( h ) C3aR expression by microglia (Iba-1 + ) in the hippocampus. Quantification of C3aR/Iba-1 colocalization (% of YF) is shown. n = 4/group. Data are presented as mean (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA); # p < 0.05, ## p < 0.01 (unpaired t-test)

Article Snippet: Cryo-cut coronal brain sections (30 μm thickness) were sequentially immersed in antigen-retrieval solution (Abcam) for 10 min; in mouse-on-mouse blocking reagent (Vector Laboratories) for 1 h; in 5% goat or donkey serum, 0.3% Triton X-100, and 0.5% bovine serum albumin for 10 min; and then incubated overnight at 4℃ with the following primary antibodies: Iba-1 (1:1,000; FUJIFILM Wako Pure Chemical Corporation, #019-19741), Iba-1 (1:1000; NOVUS Biologicals, #NB100-1028), CD68 (1:200; Bio-Rad, #MCA1957), C3aR (1:50; Santa Cruz Biotechnology, #sc-133,172), and p-mTOR (Ser2448, 1:200; Cell Signaling Technology, #5336).

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging

doi: 10.1186/s12974-024-03130-7

Figure Lengend Snippet: Microglial C3a – C3aR signaling promotes glycolysis and phagocytosis. Young microglia (postnatal day 0–2) were treated with 10 nM recombinant mouse C3a. ( a - d ) p-AKT (Ser473), p-mTOR (Ser2448) and HIF1α were assessed by Western blotting at the indicated timepoints (0–24 h), and expression was normalized to total AKT, total mTOR and β-actin, respectively. n = 5 replicates. Data are presented as mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA). ( e - g ) Seahorse assays were used to evaluate real-time glycolytic rate in young microglia (postnatal day 0–2, pooled male and female) after 18 h in vitro treatment with 10 nM recombinant mouse C3a. Stimuli were added as indicated ( e ), and basal glycolysis ( f ) and compensatory glycolysis ( g ) were determined by calculating the glycolytic Proton Efflux Rate (e; glycoPER). n = 5/group. # p < 0.05 (unpaired t-test). ( h , i ) Flow cytometry analysis was performed to assess phagocytosis of FITC-fAβ 1−42 . Rapamycin (50 µM) or 2-DG (5 mM) were pre- (1 h) and co-treated (18 h) with C3a. Proportion of phagocytic cells ( h ) was assessed by evaluating FITC-positive microglia, and FITC MFI was evaluated in total live microglia ( i ). Data are presented as mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA)

Article Snippet: Cryo-cut coronal brain sections (30 μm thickness) were sequentially immersed in antigen-retrieval solution (Abcam) for 10 min; in mouse-on-mouse blocking reagent (Vector Laboratories) for 1 h; in 5% goat or donkey serum, 0.3% Triton X-100, and 0.5% bovine serum albumin for 10 min; and then incubated overnight at 4℃ with the following primary antibodies: Iba-1 (1:1,000; FUJIFILM Wako Pure Chemical Corporation, #019-19741), Iba-1 (1:1000; NOVUS Biologicals, #NB100-1028), CD68 (1:200; Bio-Rad, #MCA1957), C3aR (1:50; Santa Cruz Biotechnology, #sc-133,172), and p-mTOR (Ser2448, 1:200; Cell Signaling Technology, #5336).

Techniques: Recombinant, Western Blot, Expressing, In Vitro, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging

doi: 10.1186/s12974-024-03130-7

Figure Lengend Snippet: Altered expression of complement pathway components by old hippocampal microglia, especially in females. ( a ) Complement System pathway identified by performing IPA analysis of old versus young female, old versus young male, and old female versus old male microglia. Dashed line: -log 10 (p-value) cutoff of 1.3 ( p < 0.05). ( b ) Expression of the complement pathway genes C3 , C3ar1 , Cd55 and Ctsl . Data are presented as mean (SD) FPKM values. n = 5/group. ( c ) Complement gene expression by homeostatic (young and old) microglia and old DAM . Dot size shows the percentage of cells expressing the genes, and the color intensity scale indicates average gene expression by all cells in the cluster. ( d , e ) Complement gene expression by WT and 5XFAD homeostatic microglia and 5XFAD DAM from male and female mice (1 male and 2 females; d ), and homeostatic microglia and DAM1 and DAM2 subsets (1 male and 2 females combined; e ) . ( f , g ) C3aR protein expression in the hippocampus of young and old, male and female mice. Representative immunoblot images ( f ; arrow: C3aR band) and quantification of C3aR expression ( g ) are shown. n = 5/group. Data are presented as mean (SD). ( h ) C3aR expression by microglia (Iba-1 + ) in the hippocampus. Quantification of C3aR/Iba-1 colocalization (% of YF) is shown. n = 4/group. Data are presented as mean (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA); # p < 0.05, ## p < 0.01 (unpaired t-test)

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C with anti-p-AKT (Ser 473, 1:2,000; Cell Signaling Technology, #4060), anti-AKT (1:2,000; Cell Signaling Technology, #4691), anti-p-mTOR (Ser2448, 1:2,000; Cell Signaling Technology, #5336), anti-mTOR (1:2,000; Cell Signaling Technology, #2972), anti-HIF-1α (1:2,000; Novus, #NB100-449), anti-C3aR (1:1,000, Santa Cruz, #sc-133,172), and anti-β-actin (1:1,000; Cell Signaling Technology, #3700) antibodies.

Techniques: Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging

doi: 10.1186/s12974-024-03130-7

Figure Lengend Snippet: Microglial C3a – C3aR signaling promotes glycolysis and phagocytosis. Young microglia (postnatal day 0–2) were treated with 10 nM recombinant mouse C3a. ( a - d ) p-AKT (Ser473), p-mTOR (Ser2448) and HIF1α were assessed by Western blotting at the indicated timepoints (0–24 h), and expression was normalized to total AKT, total mTOR and β-actin, respectively. n = 5 replicates. Data are presented as mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA). ( e - g ) Seahorse assays were used to evaluate real-time glycolytic rate in young microglia (postnatal day 0–2, pooled male and female) after 18 h in vitro treatment with 10 nM recombinant mouse C3a. Stimuli were added as indicated ( e ), and basal glycolysis ( f ) and compensatory glycolysis ( g ) were determined by calculating the glycolytic Proton Efflux Rate (e; glycoPER). n = 5/group. # p < 0.05 (unpaired t-test). ( h , i ) Flow cytometry analysis was performed to assess phagocytosis of FITC-fAβ 1−42 . Rapamycin (50 µM) or 2-DG (5 mM) were pre- (1 h) and co-treated (18 h) with C3a. Proportion of phagocytic cells ( h ) was assessed by evaluating FITC-positive microglia, and FITC MFI was evaluated in total live microglia ( i ). Data are presented as mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA)

Article Snippet: Membranes were blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated overnight at 4 °C with anti-p-AKT (Ser 473, 1:2,000; Cell Signaling Technology, #4060), anti-AKT (1:2,000; Cell Signaling Technology, #4691), anti-p-mTOR (Ser2448, 1:2,000; Cell Signaling Technology, #5336), anti-mTOR (1:2,000; Cell Signaling Technology, #2972), anti-HIF-1α (1:2,000; Novus, #NB100-449), anti-C3aR (1:1,000, Santa Cruz, #sc-133,172), and anti-β-actin (1:1,000; Cell Signaling Technology, #3700) antibodies.

Techniques: Recombinant, Western Blot, Expressing, In Vitro, Flow Cytometry