Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos increased necroptosis and M1 polarization in hemin-induced primary murine microglia. ( A ) Cell viability was determined using the CCK-8 assay. ( B ) Cell apoptosis was assessed via flow cytometry. ( C ) Western blot analysis was conducted to measure p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL levels. ( D and E ) Flow cytometry was used to examine the proportions of CD86 + and CD163 + macrophages. ( F ) qRT-PCR analysis of iNOS, COX-2, CCL2, ARG1, CD206, and YM1 levels. ( G ) iNOS, COX-2, ARG1, and CD206 proteins levels. ( H ) ELISA was conducted to assess IL-1β, IL-6, and TNF-α levels. n = 3. * p < 0.05 vs. Control group; # p < 0.05 vs. Hemin group

Article Snippet: The mouse TNF-α (CSB-E04741m, CUSABIO, China), human TNF-α (KE00154, Proteintech), IL-1β (KE10003, KE00021, Proteintech), IL-6 (CSB-E04639m, CSB-E04638h, CUSABIO), and C5a (CSB-E08512h, CUSABIO) ELISA kits were operated according to the manufacturer’s instructions.

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos increased necroptosis of macrophages/microglia and M1 polarization after ICH. ( A ) Cell apoptosis was detected by TUNEL. ( B ) p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL protein levels. ( C and D ) Immunofluorescence was used to detect IBA1 + iNOS + and IBA1 + ARG1 + . ( E ) iNOS, COX-2, CCL2, ARG1, CD206, and YM1 mRNA levels. ( F ) iNOS, COX-2, ARG1, and CD206 levels were analyzed by western blot. ( G ) ELISA was conducted to examine IL-1β, IL-6, and TNF-α. n = 5. * p < 0.05 vs. Sham group; # p < 0.05 vs. ICH group

Article Snippet: The mouse TNF-α (CSB-E04741m, CUSABIO, China), human TNF-α (KE00154, Proteintech), IL-1β (KE10003, KE00021, Proteintech), IL-6 (CSB-E04639m, CSB-E04638h, CUSABIO), and C5a (CSB-E08512h, CUSABIO) ELISA kits were operated according to the manufacturer’s instructions.

Techniques: TUNEL Assay, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos C5 promoted necroptosis and M1 polarization in hemin-induced microglia. ( A ) The heat map shows the differentially expressed proteins. ( B ) C5a level in brain tissue analyzed by IHC. * p < 0.05 vs. sham group. ( C ) C5a level in serum was detected by ELISA. * p < 0.05 vs. sham group. Under RIPA lysis conditions, the results of Western blot for C5a in ( D ) human RBC-Exos and ( E ) murine RBC-Exos after untreated EVs or after treatment with proteinase K (5 U/mL, 10 min) . # p < 0.05 vs. Sham-Exos/Normal-Exos group; @ p < 0.05 vs. ICH-Exos group. ( F ) C5a level in HMC3 cells was analyzed by Western blot and ELISA. ( G ) Cell viability was detected by CCK-8. ( H ) Cell apoptosis was detected by flow cytometry. ( I ) p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL levels were detected by Western blot. ( G ) The proportions of CD86 + M1 and CD163 + M2 macrophages were detected by flow cytometry. ( K ) iNOS, COX-2, CCL2, ARG1, CD206, and YM1 levels analyzed by qRT-PCR. ( L ) iNOS, COX-2, ARG1 and CD206 levels analyzed by Western blot. (M) ELISA was used to detect IL-1β, IL-6, and TNF-α levels. n = 3. # p < 0.05 vs. Normal-Exos group; & p < 0.05 vs. ICH-Exos group

Article Snippet: The mouse TNF-α (CSB-E04741m, CUSABIO, China), human TNF-α (KE00154, Proteintech), IL-1β (KE10003, KE00021, Proteintech), IL-6 (CSB-E04639m, CSB-E04638h, CUSABIO), and C5a (CSB-E08512h, CUSABIO) ELISA kits were operated according to the manufacturer’s instructions.

Techniques: Paraffin-embedded Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Lysis, Western Blot, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Chromatin accessibility analysis identifies the transcription factor ETV5 as a suppressor of adipose tissue macrophage activation in obesity

doi: 10.1038/s41419-021-04308-0

Figure Lengend Snippet: A BMDMs were transfected with siRNA negative control (siRNA NC) or siRNA specific to Etv5 (siRNA Etv5 ) 48 h and Il6 expression was assessed by RT-qPCR 48 h later. Il6 expression levels of Raw264.7 cells after knockdown of Etv5 using shRNA B or overexpression of Etv5 C . Il6 expression levels of Raw264.7 cells after knockdown of Etv5 using shRNA D or overexpression of Etv5 E under MMe induction condition. F Il6 expression levels of BMDMs after treatment with palmitate, glucose, and insulin alone or with a combination of these compounds for 24 h. G–I Measuring IL-6 protein levels in the supernatant of Raw264.7 cell culture by ELISA. Cells were treated with palmitate (P) or PGI G , or underwent Etv5 knockdown using siRNA H , or shRNA I . Data in this result were all representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t test.

Article Snippet: Supernatants were collected after macrophage culture and IL-6 protein concentrations were measured using a mouse IL-6 ELISA kit (E-MSEL-M0001, Elabscience, China) according to the manufactories’ instructions.

Techniques: Transfection, Negative Control, Expressing, Quantitative RT-PCR, Knockdown, shRNA, Over Expression, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: A mutation in Irak2c identifies IRAK-2 as a central component of the TLR regulatory network of wild-derived mice

doi: 10.1084/jem.20090490

Figure Lengend Snippet: TLR2-stimulated IL-6 production is associated with the Why1 locus. (A) Codominant inheritance of IL-6 protein secretion. Peritoneal macrophages from parental strains and F1(C57BL/6JxMOLF/Ei) hybrid mice were plated and stimulated for 6 h with the indicated concentrations of LTA. IL-6 secretion was measured by ELISA. Results are ± SEM from triplicate wells, representative of three independent experiments. (B) Peritoneal macrophages from 82 N2 (C57BL/6Jx(C57BL/6JxMOLF/Ei)) mice were plated and stimulated with 2 µg/ml LTA as in A. Mean IL-6 production from triplicate wells is plotted for each mouse, grouped by genotype at D6MIT328, a marker near the peak of the Why1 locus. (C) Peritoneal macrophages from 50 independent N2 backcross mice were stimulated for 1 h with 2 µg/ml LTA. IL-6 mRNA was quantified by real time PCR. Mean mRNA production from two wells is plotted, grouped by genotype at D6MIT328. P-values were calculated in B and C using a two-tailed Student's t test.

Article Snippet: IL-6 was measured using anti–mouse Duo-Set IL-6 ELISA (DY-406; R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Marker, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: A mutation in Irak2c identifies IRAK-2 as a central component of the TLR regulatory network of wild-derived mice

doi: 10.1084/jem.20090490

Figure Lengend Snippet: Increased IL-6 production in C57BL/6J- Why1 MOLF/MOLF congenic mice. (A and B) Peritoneal macrophages were isolated from wild-type and Why1 congenic mice and stimulated with 2 µg/ml LTA. IL-6 or TNF mRNA was analyzed by real-time PCR at the indicated times (A) and IL-6 protein secretion was analyzed by ELISA 6 h after stimulation (B). (C) Cells were stimulated for 2 h with 100 ng/ml LPS, 2 µg/ml LTA, 200 nM CpG, 100 µM Loxoribine, or 25 µg/ml poly(I:C), and IL-6 mRNA was analyzed by real-time PCR. Data shown as mean ± range of duplicate wells (A and C) or ± SEM of triplicate wells (B). (D) Cells from wild-type and Why1 congenic mice were stimulated with 2 µg/ml LTA for the indicated times and protein was extracted for Western blot analysis of p38 activation and IκBα degradation. Total p38 served as a loading control. (E) Peritoneal macrophages from the indicated strains were stimulated with 2 µg/ml LTA for the indicated times and CCL3 mRNA was measured by real-time PCR analysis. Error bars indicate SEM. All experiments are representative of two to three independent trials.

Article Snippet: IL-6 was measured using anti–mouse Duo-Set IL-6 ELISA (DY-406; R&D Systems).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: A mutation in Irak2c identifies IRAK-2 as a central component of the TLR regulatory network of wild-derived mice

doi: 10.1084/jem.20090490

Figure Lengend Snippet: IRAK-2 is required for early TLR2 responses in MOLF/Ei but not C57BL/6J macrophages. (A–C) C57BL/6J and MOLF/Ei BMDMs were infected with lentiviral constructs containing either control or one of three IRAK-2–targeting shRNA sequences. (A) To assess knockdown efficiency, mRNA was harvested and cDNA was amplified with IRAK-2 or GAPDH-specific PCR primers. (B and C) Cells were stimulated with 2 µg/ml LTA and IL-6 was quantified at 4 h using RT-PCR, with error bars indicating SEM (B), or at 6 h using ELISA, with error bars indicating SEM (C). n.d., not detected. Data are representative of three independent experiments. (D and E) MOLF/Ei BMDMs were infected with control or IRAK-2–targeting construct number two and stimulated with 2 µg/ml LTA (D) or under hypertonic NaCl conditions (E) for the indicated times. MAPK and NF-κB pathway signaling events were assessed using the indicated Western blot analyses. Total p38 and ERK serve as loading controls. Data are representative of at least two independent experiments.

Article Snippet: IL-6 was measured using anti–mouse Duo-Set IL-6 ELISA (DY-406; R&D Systems).

Techniques: Infection, Construct, Control, shRNA, Knockdown, Amplification, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: A mutation in Irak2c identifies IRAK-2 as a central component of the TLR regulatory network of wild-derived mice

doi: 10.1084/jem.20090490

Figure Lengend Snippet: The effect of IRAK-2 knockdown in macrophages is allele specific. (A and B) BMDMs from C57BL/6J and C57BL/6J- Why1 MOLF/MOLF mice were transduced with control or an IRAK-2–targeting shRNA construct. Cells were stimulated with 2 µg/ml LTA for 6 h followed by IL-6 ELISA analysis (A) or for the indicated times followed by IL-6 mRNA quantification by real-time PCR (B). Real-time PCR data are shown as the fold change (in Ct values) observed in IRAK-2 shRNA treated relative to control-treated cells. Data shown as mean ± SEM from triplicate wells (A) or ± range from duplicate wells (B), representative of two independent experiments. (C) Peritoneal macrophages from 14 Why1 homozygous or 9 heterozygous N2 backcross mice were plated and infected with control or IRAK-2–targeting shRNA constructs. 3 d after infection, cells were stimulated with 2 µg/ml LTA and IL-6 protein secretion was measured by ELISA. Mean values of three wells for each mouse are plotted. P-values were calculated using a paired Student's t test analysis.

Article Snippet: IL-6 was measured using anti–mouse Duo-Set IL-6 ELISA (DY-406; R&D Systems).

Techniques: Knockdown, Transduction, Control, shRNA, Construct, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Infection