inos polyclonal antibody  (Proteintech)

Journal: Bioactive Materials

Article Title: Geometry-driven immunomodulation in 3D-printed bioceramics: Negative curvature promotes macrophage M2 polarization via Ras-MAPK/HIF-1α signaling for vascularized osteogenesis

doi: 10.1016/j.bioactmat.2026.01.001

Figure Lengend Snippet: Macrophage polarization analysis of Raw264.7 on structures with different gaussian curvature: (A, B) Chord Diagram for qPCR analysis of CCR7, IL6, iNOS-inflammatory and M1 marker genes, and Arg-1, CD206, IL10-M2 related protein genes in different Gaussian curvature groups. (C) Protein content of Arg-1 in different Gaussian curvature groups at 1 and 3 days. (D) Integral plots of the five experimental groups. IL4 group is the positive control for CD206 expression and lipopolysaccharide (LPS) group is the negative control.

Article Snippet: The reagents used in the experiment included: H-DMEM(11965118, Gibco, USA.), α-DMEM medium(12571063, Gibco, USA.), TritonX-100(ST1723, Beyotime, China), 4 % paraformaldehyde (BL539A, Biosharp, China),FBS(A5256701, Gibco, USA.),ECM medium (Science Cell, USA.),and DAPI staining solution (C1006, Beyotime, China),BCIP/NBT(C3206, Beyotime, China), reactive oxygen species kit (S0033S, Beyotime, China), BSA (B2064, ≥98 %, Sigma-Aldrich, USA.),CD31 antibody (ab28364, Abcam, USA.), secondary anti-IGg (ab175773, Alexa Fluor® 680, Abcam, USA.), Phalloidin-iFluor 488(ab176753, Abcam, USA.), CCR7(AF5293, Bioss, China), CD206 (bsm-60761R, Bioss, China), iNOS (bs-22924R, Bioss, China), RIPA (P0013, Beyotime, China), p-ERK1/2 (AF3687, Affinity, USA.) and ERK1/2 (#AF0155, Affinity, USA), luminol detection reagent (sc-2048, Santa Cruz, USA.), GAPDH (Cat#KC-5G5, Kangchen Biotechnology, China), Trizol(15596026CN, Invitrogen, USA.), DEPC(R0601, Thermo Scientific, USA.), TBST(R017R.0000, Thermo Scientific, USA.), HIF-1a(GTX127309, GeneTex, USA.), β-Tubulin(10094-1-AP, Proteintech, UK) Adezmapimod (SB 203580, MCE, USA.) medium and Paclitaxel (99.88 %, HY-B0015R, MCE), ELISA Arg-1(E-EL-M3092, ELabSci@, China), TNF-α (E-EL-M3063, ELabSci@, China), OPN(22952-1-AP, Proteintech, UK.), F4/80 (GB11027-100, Servicebio, China) Alkaline phosphatase activity kit (P0321S, Beyotime, China), Matrigel (CLS356234, Corning, USA), Microfill MV120 (Flow tech, USA), EDTA(17892, Thermo Scientific, USA.) xylene(X112051, AR,99 %, Aladdin, China), ethanol (107-21-1, AR,99 %, Aladdin, China).

Techniques: Marker, Positive Control, Expressing, Negative Control

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate-mediated modulation of macrophage activity alters endometriotic lesion p rogression (A) mRNA expression of IL1B , IL6 , TNFA , and iNOS in peritoneal macrophages from endometriosis (EM) and non-EM patients, as well as non-EM macrophages co-cultured with either nor-ESC or ectopic ec-ESC for 12 h (n = 3/group).(B) Migration of si- Irg1 or NC-treated ectopic ESCs was assessed after co-culture with PBMCs for 48 h. Quantification of migrated cells in five random fields per group are shown (n = 3/group). (C-D) PKH67-labeled human ectopic ESCs pretreated with si- Irg1 or NC were co-cultured with PBMCs for 8 h. Phagocytosis of ESCs by macrophages was analyzed by flow cytometry with representative gating (C), PKH67 signal and quantification of PKH67-positive macrophages (D) (n = 6/group).(E,G) Flow cytometry analysis and quantification of iNOS + peritoneal macrophages from mouse model in E (E), A (G). (n = 6/group). (F,H) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages from mouse model in E (E), A (G) (n = 6/group).(I)Quantification of itaconate in endometriotic lesion tissue by LC–MS from mice treated with IRG1-IN-1 or vehicle. (n = 3/group).(J) Flow cytometry analysis and quantification of iNOS + peritoneal macrophages from mouse model in J (n = 6/group).(K,L) Migration of mESCs induced by peritoneal macrophages from PBS- or 4-OI-treated mice was assessed by transwell assay; representative images and quantification of migrated cells are shown (n = 5/group). (M) Flow cytometry analysis and quantification of phagocytosis of PKH67-labeled mESCs by peritoneal macrophages (n = 6/group).(N) Schematic of the experimental design for clodronate liposome-mediated macrophage depletion and 4-OI intervention in EM mice. (O, P) Representative images (O) and gross morphology (P) of endometriotic lesions in control, clodronate, PBS, and 4-OI groups. (Q) Quantification of lesion weight in mice treated with clodronate liposomes or control liposomes, with or without 4-OI (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) E2, estradiol benzoate; EM, endometriosis; LNP, lipid nanoparticle; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; Clod, clodronate liposome; PBS, phosphate-buffered saline; si- Irg1 , small interfering RNA targeting Irg1.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Activity Assay, Expressing, Cell Culture, Migration, Co-Culture Assay, Labeling, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy, Transwell Assay, Control, Liposomes, Saline, Small Interfering RNA

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Inhibition of NRF2 Signaling Reverses the Anti-inflammatory and Anti-endometriotic Effects of Itaconate in Macrophages and a Mouse Model of End ometriosis (A,B) Western blot analysis (A) and quantification (B) of NRF2 protein levels in bone marrow-derived macrophages (BMDMs) pretreated with LPS (100 ng/mL), 4-octyl itaconate (4-OI, 250 μM), or both for 12 h (n = 3/group). (C,D) Western blot analysis (C) and quantification (D) of NRF2 in BMDMs treated with LPS, 4-OI, and the NRF2 inhibitor ML385 (2.5 μM) for 12 h (n = 3/group). (E) Flow cytometry analysis and quantification of iNOS + BMDMs after indicated treatments (n = 3/group). (F) mRNA expression of pro-inflammatory genes ( Il1b , Il6 , Nos2 , Tnf ) in BMDMs under different conditions (n = 3/group). (G) Flow cytometry analysis and quantification of iNOS + BMDMs following NRF2 knockdown (si Nrf2 ) with or without 4-OI, compared to negative control (NC) (n = 3/group).(H) Schematic of the experimental design for ML385 administration in a mouse model of endometriosis. (I) Representative images of endometriotic lesions in PBS- and ML385-treated mice (lesions marked by red circles). (J) Gross morphology of lesions in both groups(n = 6/group). (K) Quantification of lesion weight (n = 6/group). (L) Flow cytometry analysis and quantification of iNOS + peritoneal macrophages from EM mice treated with PBS or ML385 (n = 6/group). (M) mRNA expression of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages from each group (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) E2, estradiol benzoate; EM, endometriosis; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; ML385, NRF2 inhibitor.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Inhibition, Western Blot, Derivative Assay, Flow Cytometry, Expressing, Knockdown, Negative Control

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate Regulates Lysosomal Function, Calcium Signaling, and p38 MAPK Pathway in Macrophages (A) Lysosomal acidification in ascites-derived macrophages from non-endometriosis (NC) and endometriosis (EM) patients assessed by LysoSensor fluorescence intensity (n = 6/group). (B) Lysosomal acidification in PBMC-derived macrophages after LPS,IL-4 and 4-OI treatment (n = 3/group). (C) Lysosomal pH value of BMDMs after LPS and 4-OI treatment (n = 3/group). (D, E) Effect of chloroquine (CQ) on LysoSensor intensity (D) and proportion of iNOS + BMDMs (E) after LPS and 4-OI treatment (n = 3/group). (F) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in BMDMs under indicated treatments (n = 3/group). (G) Intracellular Ca 2+ dynamics in BMDMs after treatments, measured by Fluo-4. (H) Intracellular Ca 2+ dynamics in ascites-derived macrophages from NC and EM patients. (I) Quantification of intracellular calcium in peritoneal macrophages from NC and EM patients (n = 3/group). (J) Quantification of intracellular calcium in BMDMs (n = 5/group). (K) mRNA expression of lysosomal calcium channel genes ( MCOLN1 , MCOLN2 , TPC1 , TPC2 ) in ascites-derived macrophages from NC and EM patients (n = 3/group). (L) lysosomal calcium channel genes mRNA in PBMC-derived macrophages co-cultured with normal ESCs (Nor-ESC), eutopic ESCs (Eu-ESC), or ectopic ESCs (Ec-ESC) from patients (n = 5/group). (M − N) Fluo-4-based Ca 2+ dynamics in BMDMs treated with LPS, 4-OI, ML-SA1 (M), or CQ (N). (O) Flow cytometry analysis and quantification of iNOS + BMDMs after indicated treatments (n = 3/group). (P) mRNA levels of pro-inflammatory genes in BMDMs under different treatments (n = 3/group). (Q) Western blot and quantification of p-p38 and total p38 in BMDMs with LPS ± 4-OI (n = 3/group). (R–S) Western blot and quantification of iNOS, p-p38, and total p38 in BMDMs treated with LPS, 4-OI, and CQ (R), or ML-SA1 (S) (n = 3/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) EM, endometriosis; NC, non-EM control; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; CQ, chloroquine; ML-SA1, MCOLN channel agonist; PBMC, peripheral blood mononuclear cell; ESC, endometrial stromal cell.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Derivative Assay, Fluorescence, Expressing, Cell Culture, Flow Cytometry, Western Blot, Control

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate Suppresses NOX2-Derived ROS to Regulate Macrophage Function and Lesion Progression in End ometriosis (A-B) qPCR analysis of NOX2 mRNA in peritoneal macrophages (PMs) from human (A) and mouse (B) NC and EM groups (n = 3/group). (C-D) Western blot and quantification of NOX2 protein in PBMC-derived macrophages treated with LPS or LPS + 4-OI (n = 5/group). (E) NOX2 enzyme activity in BMDMs (n = 4/group). (F, G) Flow cytometry and quantification of ROS production in BMDMs after LPS or LPS + 4-OI (n = 3/group). (H, I) Flow cytometry and quantification of iNOS + BMDMs after LPS, LPS + 4-OI, or LPS + 4-OI + DPI treatment (n = 4/group). (J) Intracellular Ca 2+ dynamics in BMDMs measured by Fluo-4 after LPS, 4-OI, or DPI treatment. (K) Schematic of 4-OI and DPI intervention in the mouse endometriosis model. (L, M) Representative images (L) and gross morphology (M) of endometriotic lesions after PBS, 4-OI, or DPI treatment. (N) Quantification of lesion weight (n = 6/group). (O, P) Flow cytometry and quantification of iNOS + peritoneal macrophages (percentage and MFI) in peritoneal lavage (n = 6/group). (Q) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages after treatments (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns: not significant.) E2, estradiol benzoate; EM, endometriosis; NC, non-EM control; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; DPI, NOX2 inhibitor.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Derivative Assay, Western Blot, Activity Assay, Flow Cytometry, Control