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Cell Signaling Technology Inc anti tnf α d2d4
Anti Tnf α D2d4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/d2d4/pm41893475-97-30-39
Average 86 stars, based on 1 article reviews

anti tnf α d2d4 - by Bioz Stars, 2026-09

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Article Title: Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation
Article Snippet: Cleaved Caspase-9 (Asp330) , D2D4 , rabbit , Cell Signaling Technology , CST-7237 , 1:1000.

Article Title: Caspase-independent programmed cell death triggers Ca 2 PO 4 deposition in an in vitro model of nephrocalcinosis
Article Snippet: Cleaved Caspase-9 (Asp 330 ) , D2D4 , Rabbit , Cell Signaling Technology , CST-7237 , 1:1000.

Article Title: Murine RAW Macrophages Are a Suitable Model to Study the CD3 Signaling in Myeloid Cells
Article Snippet: anti mouse TNF-α Rabbit Antibody , Cell Signaling , D2D4 , 11948T.

Incubation:

Article Title: 2′Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors
Article Snippet: Thirty micrograms of protein lysates was electrophoretically separated on 4%–20% Mini-PROTEAN ® TGXTM polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene fluoride membranes (PolyScreen ® ; PerkinElmer). .. After rinsing with TBST20, membranes were blocked for 1 h in 5% dry milk in TBST20, followed by overnight incubation with primary antibodies, including anti-IPS-1 (1:200) (E-3; Santa Cruz Biotechnology), anti-RIG-I (1:500) (D14G6; Cell Signaling), anti-MDA-5 (1:500) (D74E4; Cell Signaling), anti-PKR (1:350) (Catalog No 3072; Cell Signaling), anti-cleaved caspase-7 (1:1,000) (D6H1; Cell Signaling), anti-cleaved caspase-9 (1:500) (D2D4; Cell Signaling), anti-cleaved poly(ADP-ribose) polymerase (PARP) (1:1,000) (D64E10; Cell Signaling), or anti-beta-tubulin (1:1,000) (9F3; Cell Signaling). .. When different proteins were sequentially detected on the same membrane, membranes were treated for 8 min with Restore Western Blot Stripping Buffer (Thermo Scientific), washed, blocked, and probed again, as described above.

Article Title: 2-fluoro-modified RNAs as immunostimulators
Article Snippet: Twelve to 20 μg of protein lysates were electrophoretically separated on 4-20% Mini-PROTEAN® TGXTM polyacrylamide gels (Bio-Rad, Hercules, Calif.) and transferred to polyvinylidene fluoride (PVDF) membranes (PolyScreen®, PerkinElmer). .. After rinsing in TBST20, membranes were blocked for 1 h in 10% dry milk in TBTS20, followed by overnight incubation with primary antibodies anti-MAVS (1:200) (E-3; Santa Cruz), anti-RIG-I (1:500) (D14G6; Cell Signaling, Danvers, Mass.), anti-MDA5 (1:500) (D74E4; Cell Signaling), anti-PKR (1:350) (Catalog No 3072; Cell Signaling), anti-cleaved caspase-9 (1:500) (D2D4; Cell Signaling), anti-cleaved caspase-7 (1:1,000) (D6H1; Cell Signaling), and anti-cleaved PARP (1:1,000) (D64E10; Cell Signaling). .. When different proteins were sequentially detected on the same membrane, membranes were treated for 8 minutes with Restore Western Blot Stripping Buffer (Thermo Scientific), washed, blocked and probed again, as described above.

Western Blot:

Article Title: Expression of the vault RNA protects cells from undergoing apoptosis
Article Snippet: Immune complexes were visualized using an enhanced chemiluminescence (ECL) reagent, or ECL-femto (Pierce Thermo scientific) or were visualized with the Odyssey imaging system according to the manufacturer's instructions. .. The following antibodies and dilutions for western blotting were used: mouse anti-MVP 1:500 (Santa Cruz); mouse anti-HA tag (16B12, Covance) 1:500; rabbit anti-FLAG 1:5,000 (F7425, Sigma), Histidine Tag (6xHis) mouse anti-histidine Tag monoclonal Antibody (clone 3D5, Life Technologies); rat anti-EBNA1 1:50 (ref. ); rat anti-EBNA2 (1:20, ref. ); mouse anti-EBV 1:1,000 (Dianova),rabbit anti–PDCD4 1:1,000 (D29C6, Cell Signaling); rabbit anti-cleaved caspase-3 1:1,000 (5A1E, Cell Signaling); rabbit anti-cleaved caspase-9 1:1,000 (D2D4, Cell Signaling); mouse anti-cleaved caspase-8 1:1,000 (11G10, Cell Signaling); goat anti-mouse IgG HRP Conjugate 1:10,000 (Invitrogen); goat anti–rabbit IgG HRP Conjugate 1:15,000 (Pierce); anti-ribosomal protein L9 (Santa Cruz); rabbit anti-Bcl-xL 1:1,000 (54H6, Cell Signaling), rabbit anti-ARC 1:1,000 (sc-11435, Santa Cruz); rabbit anti-GAPDH 1:1,000 (14C10, Cell Signaling), rabbit anti-NFκB p65 (Santa Cruz, sc-372) 1: 500, rabbit anti Phospho-IkBα (Ser32) 1:250 (14D4, Cell Signaling), mouse anti-HSP 90 1:1,000 (AC-16, Santa Cruz). ..

Immunohistochemistry:

Article Title: Modulation of Oxidative Stress and Inflammation in the Aged Lacrimal Gland.
Article Snippet: Secondary goat anti-rabbit Alexa-Fluor 488 conjugated IgG antibodies were used, as previously described.29 The images were captured by an Eclipse E400 Nikon fluorescence microscope equipped with a DS-F1 digital camera. .. Immunohistochemistry was performed to detect IL-1b (1:100; rabbit monoclonal clone D4T2D; Cell Signaling Technology, Danvers, MA; catalog number 12426S), MHC II (1:100; I-A/I-E; clone M5/114.15.2; BD Pharmingen, San Diego, CA; catalog number 556999), TNF-a (1:00 dilution; rabbit monoclonal antibody; clone D2D4; Cell Signaling Technology; catalog number 11948), or cathepsin S (1:00 dilution; Santa Cruz Biotechnology, Dallas, TX; catalog number sc-271619) antibodies and appropriate biotinylated secondary antibodies (BD Biosciences, San Diego, CA) and a Vectastain Elite ABC kit using NovaRed reagents (Vector Laboratories, Burlingame, CA), as previously described.30 Lymphocytic infiltration foci were counted in hematoxylin and eosinestained lacrimal gland sections by standard light microscopy using a 10 objective (Nikon; Eclipse E400) by two masked observers. ..

Immunopeptidomics:

Light Microscopy:

Image Search Results

In vivo sepsis efficacy evaluation of SAMPs. ( A ) Bacterial quantification in the MDR- E. coli -infected liver, spleen, lung, kidney, and PLF after treatment with PBS, FA, Amcill-s, or KR (5 mg kg -1 ). ( B ) Body weight changes of mice during treatment. Relative organ index (organ mass/weight of mice before execution) of ( C ) liver, ( D ) spleen, ( E ) lung, and ( F ) kidney after treatment. The ( G ) TNF-α and ( H ) IL-6 levels in mouse serum after treatment. Data are presented as mean ± SD ( n = 6); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.005. ( I ) Immunofluorescence (TNF-α and IL-6) images of liver, spleen, lung, and kidney after treatment, Scale bar: 100 μm. ( J ) Gram-stained images and ( K ) H&E-stained images of liver, spleen, lung, and kidney after treatment, Scale bar: 200 μm.

Journal: Antimicrobial Agents and Chemotherapy

Article Title: A short antimicrobial peptides family demonstrates efficacy to infection via a multimodal mechanism of action

doi: 10.1128/aac.01343-25

Figure Lengend Snippet: In vivo sepsis efficacy evaluation of SAMPs. ( A ) Bacterial quantification in the MDR- E. coli -infected liver, spleen, lung, kidney, and PLF after treatment with PBS, FA, Amcill-s, or KR (5 mg kg -1 ). ( B ) Body weight changes of mice during treatment. Relative organ index (organ mass/weight of mice before execution) of ( C ) liver, ( D ) spleen, ( E ) lung, and ( F ) kidney after treatment. The ( G ) TNF-α and ( H ) IL-6 levels in mouse serum after treatment. Data are presented as mean ± SD ( n = 6); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.005. ( I ) Immunofluorescence (TNF-α and IL-6) images of liver, spleen, lung, and kidney after treatment, Scale bar: 100 μm. ( J ) Gram-stained images and ( K ) H&E-stained images of liver, spleen, lung, and kidney after treatment, Scale bar: 200 μm.

Article Snippet: 10× PBS solution was procured from BioSharp (China); yeast extract and tryptone was obtained from Thermo Fisher (USA); Agar powder, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), Triton X-100, ampicillin sodium, kanamycin sulfate, crystal violet, PI solution, 10× DNA loading buffer, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), calcein-AM/PI Dead/Live Cell Dual Staining Kit and Annexin V-FITC Apoptosis Detection Kit were obtained from Solarbio (China); 3,3'′-dipropylthiadicarbocyanine Iodide (DiSC 3 -5) was purchased from AAT BioQuest (USA); HEPES buffer was purchased from Yuanye (China); methanol, 25% aqueous glutaraldehyde solution, absolute ethanol, sodium chloride (NaCl), and potassium chloride (KCl) were obtained from Sinopharm Chemical Reagent Co., Ltd. (China); TFE was obtained from Sigma Aldrich (USA); 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and dimethyl sulfoxide (DMSO) were obtained from J&K Chemical Technology (China); Micro-MDA Assay Reagent Kit was purchased from KeyGEN Biotech (China); Cell Counting Kit-8 (CCK8) was purchased from GlpBio (USA); Bacterial Genomic DNA Extraction Kit was purchased from TIANGEN (China); Bacteria RNA Extraction Kit, HiScript III RT SuperMix for qPCR(+gDNA wiper) and Taq Pro Universal SYBR qPCR Master Mix were obtained from Vazyme (China); 2 kb DNA ladder (100-2000 bp) was obtained from Baiaolaibo (China); Mouse TNF-α ELISA kit and Mouse IL-6 ELISA kit were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. (China); Anti-IL-6 antibody (ab233706) and goat Anti-rabbit IgG H&L (ab150078, Alexa Fluor 555) were obtained from Abcam (UK); Anti-TNF-α (D2D4) XP rabbit monoclonal antibody was obtained from Cell Signaling Technology (USA); All the mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (China) and kept in a specific pathogen-free environment.

Techniques: In Vivo, Infection, Immunofluorescence, Staining

A Western blot analysis of SNAP23 expression in SW620 cells transfected with shNC, shSNAP23, or sh+rSNAP23 plasmids. B Cells were treated with varying concentrations (2.5 µM, 10 µM, 20 µM) of oxaliplatin (OXA) for 72 hours. The inhibition rate was assessed using the SRB assay. C Real-time cell analysis using the SRB assay to determine the inhibition rates of OXA (10 µM) over time. D Western blot analysis of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression in control, SNAP23-knockdown, and SNAP23-restored SW620 cells treated with OXA (10 µM, 72 h). Graphical representation of tumor volumes ( E ) and weights ( F ) in control, SNAP23-knockdown, and SNAP23-restored HT29-induced CDX tumors in nude mice subjected to intraperitoneal injection of OXA (7.5 mg/kg) in the drinking water ( n = 5). PBS was used as a control. G Representative TUNEL and H&E staining images of paraffin-embedded subcutaneous tumor sections. Scale bars, 50 μm. H Quantification of fluorescence intensity. I ROS levels in HT29-induced CDX tumors. n = 5 mice per group. Data are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test ( B , C , E , F , H , I ).

Journal: Cell Death & Disease

Article Title: SNAP23 deficiency triggers Trim21 mitochondrial translocation to suppress TFAM-mediated oxidative metabolism and drive chemoresistance in colorectal cancer

doi: 10.1038/s41419-025-08252-1

Figure Lengend Snippet: A Western blot analysis of SNAP23 expression in SW620 cells transfected with shNC, shSNAP23, or sh+rSNAP23 plasmids. B Cells were treated with varying concentrations (2.5 µM, 10 µM, 20 µM) of oxaliplatin (OXA) for 72 hours. The inhibition rate was assessed using the SRB assay. C Real-time cell analysis using the SRB assay to determine the inhibition rates of OXA (10 µM) over time. D Western blot analysis of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression in control, SNAP23-knockdown, and SNAP23-restored SW620 cells treated with OXA (10 µM, 72 h). Graphical representation of tumor volumes ( E ) and weights ( F ) in control, SNAP23-knockdown, and SNAP23-restored HT29-induced CDX tumors in nude mice subjected to intraperitoneal injection of OXA (7.5 mg/kg) in the drinking water ( n = 5). PBS was used as a control. G Representative TUNEL and H&E staining images of paraffin-embedded subcutaneous tumor sections. Scale bars, 50 μm. H Quantification of fluorescence intensity. I ROS levels in HT29-induced CDX tumors. n = 5 mice per group. Data are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test ( B , C , E , F , H , I ).

Article Snippet: Cleaved Caspase-9 (Asp330) (D2D4) Rabbit (for WB) , CST , Cat# 7237, RRID:AB_10895832.

Techniques: Western Blot, Expressing, Transfection, Inhibition, Sulforhodamine B Assay, Cell Analysis, Control, Knockdown, Injection, TUNEL Assay, Staining, Fluorescence

A Cells were treated with varying concentrations (2.5 µM, 10 µM, 20 µM) of OXA for 72 h. Intracellular ROS levels were measured using a DCFH-DA probe via flow cytometry (FC) in parental cells. B Intracellular ROS levels at different time points were measured using a DCFH-DA probe via FC following treatment with 10 µM OXA in parental cells. C – F Parental cells were treated with PBS or OXA (10 µM, 72 h) in combination with 5 mM N-acetyl-cysteine (NAC). FC and quantification analysis ( C ) with Annexin V/PI staining were used to evaluate the percentages of live cells (Annexin V−/PI−) and apoptotic cells (Annexin V+/PI+). Cell viability ( D ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( E ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( F ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. Representative FC plot ( G ) and statistical results of ( H ) total ROS levels. I Changes in mitochondrial ROS levels, mitochondrial membrane potential (ΔΨ m) of control, SNAP23-knockdown, and SNAP23-restored SW620 cells under PBS or OXA treatment (10 µM, 72 h). I , J Control, SNAP23-knockdown, and SNAP23-restored SW620 cells were treated with OXA (10 µM, 72 h) with or without 5 mM NAC. FC and quantification analysis ( I ) with Annexin V/PI staining were used to evaluate the percentages of live cells (Annexin V−/PI−) and apoptotic cells (Annexin V+/PI+). Cell inhibition rate ( I ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( I ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( J ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. Data are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test ( A – E , H , I ).

Journal: Cell Death & Disease

Article Title: SNAP23 deficiency triggers Trim21 mitochondrial translocation to suppress TFAM-mediated oxidative metabolism and drive chemoresistance in colorectal cancer

doi: 10.1038/s41419-025-08252-1

Figure Lengend Snippet: A Cells were treated with varying concentrations (2.5 µM, 10 µM, 20 µM) of OXA for 72 h. Intracellular ROS levels were measured using a DCFH-DA probe via flow cytometry (FC) in parental cells. B Intracellular ROS levels at different time points were measured using a DCFH-DA probe via FC following treatment with 10 µM OXA in parental cells. C – F Parental cells were treated with PBS or OXA (10 µM, 72 h) in combination with 5 mM N-acetyl-cysteine (NAC). FC and quantification analysis ( C ) with Annexin V/PI staining were used to evaluate the percentages of live cells (Annexin V−/PI−) and apoptotic cells (Annexin V+/PI+). Cell viability ( D ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( E ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( F ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. Representative FC plot ( G ) and statistical results of ( H ) total ROS levels. I Changes in mitochondrial ROS levels, mitochondrial membrane potential (ΔΨ m) of control, SNAP23-knockdown, and SNAP23-restored SW620 cells under PBS or OXA treatment (10 µM, 72 h). I , J Control, SNAP23-knockdown, and SNAP23-restored SW620 cells were treated with OXA (10 µM, 72 h) with or without 5 mM NAC. FC and quantification analysis ( I ) with Annexin V/PI staining were used to evaluate the percentages of live cells (Annexin V−/PI−) and apoptotic cells (Annexin V+/PI+). Cell inhibition rate ( I ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( I ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( J ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. Data are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test ( A – E , H , I ).

Article Snippet: Cleaved Caspase-9 (Asp330) (D2D4) Rabbit (for WB) , CST , Cat# 7237, RRID:AB_10895832.

Techniques: Flow Cytometry, Staining, Sulforhodamine B Assay, Activation Assay, Caspase-3 Activity Assay, Western Blot, Expressing, Membrane, Control, Knockdown, Inhibition

A The proliferative abilities of SW620 cells were detected by EdU assays. Scale bar, 100 μm. B – E The oeNC or oeTFAM plasmids were transfected into SNAP23-silenced cells or SNAP23-stably expressing cells, respectively. These cells were treated with OXA (10 µM, 72 h). Flow cytometry (FC) ( B ) and quantification analysis ( C ) with Annexin V/PI staining were used to evaluate the percentages of live cells and apoptotic cells. Cell inhibition rate ( C ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( C ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( D ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. E Statistical results of total ROS levels. Changes in mitochondrial ROS levels, mitochondrial membrane potential (ΔΨ m). F , G TFAM-stably expressing and TFAM-overexpressing SW620 cells were treated with OXA (10 µM, 72 h) with or without 5 mM NAC. FC and quantification analysis ( F ) with Annexin V/PI staining were used to evaluate the percentages of live cells and apoptotic cells. Cell inhibition rate ( F ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( F ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( G ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. Data are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test ( C , E , F ).

Journal: Cell Death & Disease

Article Title: SNAP23 deficiency triggers Trim21 mitochondrial translocation to suppress TFAM-mediated oxidative metabolism and drive chemoresistance in colorectal cancer

doi: 10.1038/s41419-025-08252-1

Figure Lengend Snippet: A The proliferative abilities of SW620 cells were detected by EdU assays. Scale bar, 100 μm. B – E The oeNC or oeTFAM plasmids were transfected into SNAP23-silenced cells or SNAP23-stably expressing cells, respectively. These cells were treated with OXA (10 µM, 72 h). Flow cytometry (FC) ( B ) and quantification analysis ( C ) with Annexin V/PI staining were used to evaluate the percentages of live cells and apoptotic cells. Cell inhibition rate ( C ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( C ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( D ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. E Statistical results of total ROS levels. Changes in mitochondrial ROS levels, mitochondrial membrane potential (ΔΨ m). F , G TFAM-stably expressing and TFAM-overexpressing SW620 cells were treated with OXA (10 µM, 72 h) with or without 5 mM NAC. FC and quantification analysis ( F ) with Annexin V/PI staining were used to evaluate the percentages of live cells and apoptotic cells. Cell inhibition rate ( F ) of SW620 cells was measured using the SRB assay. Caspase-3 activation ( F ) was measured using an Caspase-3 Activity Assay kit. Western blot analysis ( G ) of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression. Data are means ± SD. One-way ANOVA with Tukey’s multiple comparisons test ( C , E , F ).

Article Snippet: Cleaved Caspase-9 (Asp330) (D2D4) Rabbit (for WB) , CST , Cat# 7237, RRID:AB_10895832.

Techniques: Transfection, Stable Transfection, Expressing, Flow Cytometry, Staining, Inhibition, Sulforhodamine B Assay, Activation Assay, Caspase-3 Activity Assay, Western Blot, Membrane

AS reduced the number of M1 macrophages and inhibited the secretion of proinflammatory cytokines. (A) Schematic representing the immunomodulation potential of AS in macrophages. (B) Fluorescent images and (C) the number of EdU‐positive cells (red fluorescence) demonstrating the proliferation behavior of NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (Mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. (D) Fluorescent images and (E) the number of EdU positive cells (red fluorescence) demonstrating the proliferation behavior of NG macrophages, LPS‐M1 macrophages, and AS‐treated LPS‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. (F) FCM images and (G) quantitative analysis showing the proportion of CD11c + CD86 + cells in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). (H) FCM images and (I) quantitative analysis showing the proportion of CD11c + CD86 + cells in NG macrophages, LPS‐M1 macrophages, and AS‐treated LPS‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). (J,K) qRT‐PCR results displaying the expression levels of iNOS, IL‐1β, and TNF‐α genes in macrophages with different treatments (mean ± SD; one‐way ANOVA, n = 3). (L) Fluorescent images and (M) quantitative statistics reflecting the expression of TNF‐α protein in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. (N) Fluorescent images and (O) quantitative statistics demonstrating the expression of TNF‐α protein in NG macrophages, LPS‐M1 macrophages, and AS‐treated LPS‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. The cell nucleus was stained with DAPI (blue fluorescence). Statistically significant differences between groups are indicated as follows: ***p < 0.001, and ****p < 0.0001.

Journal: Exploration

Article Title: Activating Ferroptosis of M1 Macrophages: A Novel Mechanism of Asiaticoside Encapsuled in GelMA for Anti‐Inflammation in Diabetic Wounds

doi: 10.1002/EXP.20240062

Figure Lengend Snippet: AS reduced the number of M1 macrophages and inhibited the secretion of proinflammatory cytokines. (A) Schematic representing the immunomodulation potential of AS in macrophages. (B) Fluorescent images and (C) the number of EdU‐positive cells (red fluorescence) demonstrating the proliferation behavior of NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (Mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. (D) Fluorescent images and (E) the number of EdU positive cells (red fluorescence) demonstrating the proliferation behavior of NG macrophages, LPS‐M1 macrophages, and AS‐treated LPS‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. (F) FCM images and (G) quantitative analysis showing the proportion of CD11c + CD86 + cells in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). (H) FCM images and (I) quantitative analysis showing the proportion of CD11c + CD86 + cells in NG macrophages, LPS‐M1 macrophages, and AS‐treated LPS‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). (J,K) qRT‐PCR results displaying the expression levels of iNOS, IL‐1β, and TNF‐α genes in macrophages with different treatments (mean ± SD; one‐way ANOVA, n = 3). (L) Fluorescent images and (M) quantitative statistics reflecting the expression of TNF‐α protein in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. (N) Fluorescent images and (O) quantitative statistics demonstrating the expression of TNF‐α protein in NG macrophages, LPS‐M1 macrophages, and AS‐treated LPS‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 100 µm. The cell nucleus was stained with DAPI (blue fluorescence). Statistically significant differences between groups are indicated as follows: ***p < 0.001, and ****p < 0.0001.

Article Snippet: TNF‐α (D2D4) XP rabbit mAb (11948), CD11c (N418) hamster mAb (PE conjugate, 73359), and LC3B (4108S) were sourced from Cell Signaling Technology (CST, USA).

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

Ferroptosis was responsible for AS‐induced functional downregulation of HG‐M1 macrophages. (A) Representing fluorescent images showing Fe 2+ concentration in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages. Scale bar, 10 µm. (B) Fluorescent images showing ROS levels in macrophages with the above treatments. Scale bar, 10 µm. (C) Quantitative analysis of Fe 2+ concentration in (A) (mean ± SD; one‐way ANOVA, n = 3). (D) Quantitative analysis of ROS level in (C) (mean ± SD; one‐way ANOVA, n = 3). (E) FCM images and (F) corresponding statistical analysis showing ROS levels in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (G) qRT‐PCR results displaying the expression levels of ferroptosis‐associated genes, including SLC7A11, GCLC, GSS, LPCAT3, FTH1, and GPX4, in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (H) Western blotting images and (I) quantitative band intensities showing the expression levels of SLC7A11, FTH1 and GPX4 proteins in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (J) TEM images showing the morphology deformation of mitochondria (black arrows) in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 500 nm. (K) Immunofluorescence co‐staining images and (L) corresponding relative fluorescent intensity analysis showing the relative ratio of FTH1 (green fluorescence) and TNF‐α (red fluorescence) in macrophages with above treatments (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 10 µm. The cell nucleus was dyed with DAPI (blue fluorescence). Statistically significant differences between groups are indicated as follows: ns, not significant, * p < 0.05, ***p < 0.001, and ****p < 0.0001.

Journal: Exploration

Article Title: Activating Ferroptosis of M1 Macrophages: A Novel Mechanism of Asiaticoside Encapsuled in GelMA for Anti‐Inflammation in Diabetic Wounds

doi: 10.1002/EXP.20240062

Figure Lengend Snippet: Ferroptosis was responsible for AS‐induced functional downregulation of HG‐M1 macrophages. (A) Representing fluorescent images showing Fe 2+ concentration in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages. Scale bar, 10 µm. (B) Fluorescent images showing ROS levels in macrophages with the above treatments. Scale bar, 10 µm. (C) Quantitative analysis of Fe 2+ concentration in (A) (mean ± SD; one‐way ANOVA, n = 3). (D) Quantitative analysis of ROS level in (C) (mean ± SD; one‐way ANOVA, n = 3). (E) FCM images and (F) corresponding statistical analysis showing ROS levels in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (G) qRT‐PCR results displaying the expression levels of ferroptosis‐associated genes, including SLC7A11, GCLC, GSS, LPCAT3, FTH1, and GPX4, in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (H) Western blotting images and (I) quantitative band intensities showing the expression levels of SLC7A11, FTH1 and GPX4 proteins in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (J) TEM images showing the morphology deformation of mitochondria (black arrows) in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 500 nm. (K) Immunofluorescence co‐staining images and (L) corresponding relative fluorescent intensity analysis showing the relative ratio of FTH1 (green fluorescence) and TNF‐α (red fluorescence) in macrophages with above treatments (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 10 µm. The cell nucleus was dyed with DAPI (blue fluorescence). Statistically significant differences between groups are indicated as follows: ns, not significant, * p < 0.05, ***p < 0.001, and ****p < 0.0001.

Article Snippet: TNF‐α (D2D4) XP rabbit mAb (11948), CD11c (N418) hamster mAb (PE conjugate, 73359), and LC3B (4108S) were sourced from Cell Signaling Technology (CST, USA).

Techniques: Functional Assay, Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

3‐MA antagonized AS‐activated ferritinophagy, alleviating ferroptosis of HG‐M1 macrophages. (A) Ad‐mRFP‐GFP‐LC3 double fluorescence indicator and (B) corresponding quantitative analysis showing the effect of 3‐MA‐pretreatment on autophagy flux within HG‐M1 macrophages with or without post‐treatment of AS3 (mean ± SD; one‐way ANOVA, n = 5). Scale bar, 10 µm. (C) Western blotting images and (D) quantitative band intensities indicating the expression levels of autophagy‐associated proteins (ATG7, p62, ATG5, and LC‐3) and ferroptosis‐associated proteins (FTH1 and GPX4) in HG‐M1 macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (E) Representing fluorescent images showing Fe 2+ concentration inside HG‐M1 macrophages with the above treatments. Scale bar, 5 µm. (F) Representing fluorescent images of ROS level inside HG‐M1 macrophages with above treatments. Scale bar, 5 µm. (G) Quantitative analysis of Fe 2+ concentration in (E) (mean ± SD; one‐way ANOVA, n = 3). (H) Quantitative analysis of ROS level in (F) (mean ± SD; one‐way ANOVA, n = 3). (I) Fluorescent images of EdU assay (red fluorescence) showing the proliferation behavior of macrophages with the above treatments. Scale bar, 100 µm. (J) Fluorescent images reflecting TNF‐α expression levels in macrophages with above treatments. The cell nucleus was dyed with DAPI (blue fluorescence). Scale bar, 100 µm. (K) Quantitative result of EdU assay in (I) (mean ± SD; one‐way ANOVA, n = 3). (L) Quantitative analysis reflecting TNF‐α expression levels in (J) (mean ± SD; one‐way ANOVA, n = 3). Statistically significant differences between groups are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Journal: Exploration

Article Title: Activating Ferroptosis of M1 Macrophages: A Novel Mechanism of Asiaticoside Encapsuled in GelMA for Anti‐Inflammation in Diabetic Wounds

doi: 10.1002/EXP.20240062

Figure Lengend Snippet: 3‐MA antagonized AS‐activated ferritinophagy, alleviating ferroptosis of HG‐M1 macrophages. (A) Ad‐mRFP‐GFP‐LC3 double fluorescence indicator and (B) corresponding quantitative analysis showing the effect of 3‐MA‐pretreatment on autophagy flux within HG‐M1 macrophages with or without post‐treatment of AS3 (mean ± SD; one‐way ANOVA, n = 5). Scale bar, 10 µm. (C) Western blotting images and (D) quantitative band intensities indicating the expression levels of autophagy‐associated proteins (ATG7, p62, ATG5, and LC‐3) and ferroptosis‐associated proteins (FTH1 and GPX4) in HG‐M1 macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). (E) Representing fluorescent images showing Fe 2+ concentration inside HG‐M1 macrophages with the above treatments. Scale bar, 5 µm. (F) Representing fluorescent images of ROS level inside HG‐M1 macrophages with above treatments. Scale bar, 5 µm. (G) Quantitative analysis of Fe 2+ concentration in (E) (mean ± SD; one‐way ANOVA, n = 3). (H) Quantitative analysis of ROS level in (F) (mean ± SD; one‐way ANOVA, n = 3). (I) Fluorescent images of EdU assay (red fluorescence) showing the proliferation behavior of macrophages with the above treatments. Scale bar, 100 µm. (J) Fluorescent images reflecting TNF‐α expression levels in macrophages with above treatments. The cell nucleus was dyed with DAPI (blue fluorescence). Scale bar, 100 µm. (K) Quantitative result of EdU assay in (I) (mean ± SD; one‐way ANOVA, n = 3). (L) Quantitative analysis reflecting TNF‐α expression levels in (J) (mean ± SD; one‐way ANOVA, n = 3). Statistically significant differences between groups are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: TNF‐α (D2D4) XP rabbit mAb (11948), CD11c (N418) hamster mAb (PE conjugate, 73359), and LC3B (4108S) were sourced from Cell Signaling Technology (CST, USA).

Techniques: Fluorescence, Western Blot, Expressing, Concentration Assay, EdU Assay

Downregulation of FPN1 protein was also involved in AS‐induced ferroptosis of HG‐M1 macrophages. (A) Schematic representation showing that AS blocked the transportation of free Fe 2+ to out of HG‐M1 macrophages by downregulating the expression of FPN1 protein. (B) Western blotting images and (C) quantitative band intensities showing the expression level of FPN1 protein in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). (D) Immunofluorescence co‐staining of TNF‐α and FPN1 proteins and (E) corresponding analysis of relative fluorescent intensity in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 20 µm. (F) Representative fluorescent images of Fe 2+ accumulation in HG‐M1 macrophages with different groups. Scale bar, 50 µm. (G) Representative fluorescent images of ROS accumulation in HG‐M1 macrophages exposed to different groups. Scale bar, 20 µm. (H) Corresponding quantitative analysis of Fe 2+ accumulation in (F). (I) Corresponding quantitative analysis of ROS accumulation in (G) (mean ± SD; one‐way ANOVA, n = 3). (J) Relative cell viability of HG‐M1 macrophages exposed to different groups (mean ± SD; one‐way ANOVA, n = 6). (K) Quantitative analysis and (L) FCM results showing the proportion of CD11c + CD86 + cells in HG‐M1 macrophages exposed to different groups (mean ± SD; one‐way ANOVA, n = 3). The cell nucleus was dyed with DAPI (blue fluorescence). Statistically significant differences between groups are indicated as follows: ns, not significant, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Journal: Exploration

Article Title: Activating Ferroptosis of M1 Macrophages: A Novel Mechanism of Asiaticoside Encapsuled in GelMA for Anti‐Inflammation in Diabetic Wounds

doi: 10.1002/EXP.20240062

Figure Lengend Snippet: Downregulation of FPN1 protein was also involved in AS‐induced ferroptosis of HG‐M1 macrophages. (A) Schematic representation showing that AS blocked the transportation of free Fe 2+ to out of HG‐M1 macrophages by downregulating the expression of FPN1 protein. (B) Western blotting images and (C) quantitative band intensities showing the expression level of FPN1 protein in NG macrophages, HG‐M1 macrophages, and AS‐treated HG‐M1 macrophages (mean ± SD; one‐way ANOVA, n = 3). (D) Immunofluorescence co‐staining of TNF‐α and FPN1 proteins and (E) corresponding analysis of relative fluorescent intensity in macrophages with the above treatments (mean ± SD; one‐way ANOVA, n = 3). Scale bar, 20 µm. (F) Representative fluorescent images of Fe 2+ accumulation in HG‐M1 macrophages with different groups. Scale bar, 50 µm. (G) Representative fluorescent images of ROS accumulation in HG‐M1 macrophages exposed to different groups. Scale bar, 20 µm. (H) Corresponding quantitative analysis of Fe 2+ accumulation in (F). (I) Corresponding quantitative analysis of ROS accumulation in (G) (mean ± SD; one‐way ANOVA, n = 3). (J) Relative cell viability of HG‐M1 macrophages exposed to different groups (mean ± SD; one‐way ANOVA, n = 6). (K) Quantitative analysis and (L) FCM results showing the proportion of CD11c + CD86 + cells in HG‐M1 macrophages exposed to different groups (mean ± SD; one‐way ANOVA, n = 3). The cell nucleus was dyed with DAPI (blue fluorescence). Statistically significant differences between groups are indicated as follows: ns, not significant, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: TNF‐α (D2D4) XP rabbit mAb (11948), CD11c (N418) hamster mAb (PE conjugate, 73359), and LC3B (4108S) were sourced from Cell Signaling Technology (CST, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Gel‐AS activated ferroptosis of M1 macrophages in vivo. (A) Immunofluorescent staining images of F4/80 + CD86 + cells (indicated by white arrows) at wounds treated with IC, ASC, Gel, and Gel‐AS on D7. Scale bar, 200 µm. (B) Immunofluorescence staining of TNF‐α + cells at wounds with different treatments on D7. Scale bar, 500 µm. (C) Immunofluorescent staining images of F4/80 + CD206 + cells (white arrows) at wounds with different treatments on D7. Scale bar, 200 µm. (D) Immunofluorescence staining of F4/80 + FTH1 + cells (white arrows) at wounds with different treatments on D7. Scale bar, 200 µm. (E) Immunofluorescence staining of FPN1 at wounds with different treatments on D7. Cell nucleus was dyed with DAPI (blue fluorescence). Scale bar, 200 µm. (F) Corresponding quantitative statistics of F4/80 + CD86 + cells in (A) (mean ± SD; one‐way ANOVA, n = 6). (G) Quantitative analysis of TNF‐α in (B) (mean ± SD; one‐way ANOVA, n = 6). (H) Corresponding quantitative statistics of F4/80 + CD206 + cells in (C) (mean ± SD; one‐way ANOVA, n = 6). (I) Quantitative analysis of FTH1 in (D) (mean ± SD; one‐way ANOVA, n = 6). (J) Quantitative analysis of FPN1 in (E) (mean ± SD; one‐way ANOVA, n = 6). Statistically significant differences between groups are indicated as follows: ns, not significant, * p < 0.05 and ***p < 0.001.

Journal: Exploration

Article Title: Activating Ferroptosis of M1 Macrophages: A Novel Mechanism of Asiaticoside Encapsuled in GelMA for Anti‐Inflammation in Diabetic Wounds

doi: 10.1002/EXP.20240062

Figure Lengend Snippet: Gel‐AS activated ferroptosis of M1 macrophages in vivo. (A) Immunofluorescent staining images of F4/80 + CD86 + cells (indicated by white arrows) at wounds treated with IC, ASC, Gel, and Gel‐AS on D7. Scale bar, 200 µm. (B) Immunofluorescence staining of TNF‐α + cells at wounds with different treatments on D7. Scale bar, 500 µm. (C) Immunofluorescent staining images of F4/80 + CD206 + cells (white arrows) at wounds with different treatments on D7. Scale bar, 200 µm. (D) Immunofluorescence staining of F4/80 + FTH1 + cells (white arrows) at wounds with different treatments on D7. Scale bar, 200 µm. (E) Immunofluorescence staining of FPN1 at wounds with different treatments on D7. Cell nucleus was dyed with DAPI (blue fluorescence). Scale bar, 200 µm. (F) Corresponding quantitative statistics of F4/80 + CD86 + cells in (A) (mean ± SD; one‐way ANOVA, n = 6). (G) Quantitative analysis of TNF‐α in (B) (mean ± SD; one‐way ANOVA, n = 6). (H) Corresponding quantitative statistics of F4/80 + CD206 + cells in (C) (mean ± SD; one‐way ANOVA, n = 6). (I) Quantitative analysis of FTH1 in (D) (mean ± SD; one‐way ANOVA, n = 6). (J) Quantitative analysis of FPN1 in (E) (mean ± SD; one‐way ANOVA, n = 6). Statistically significant differences between groups are indicated as follows: ns, not significant, * p < 0.05 and ***p < 0.001.

Article Snippet: TNF‐α (D2D4) XP rabbit mAb (11948), CD11c (N418) hamster mAb (PE conjugate, 73359), and LC3B (4108S) were sourced from Cell Signaling Technology (CST, USA).

Techniques: In Vivo, Staining, Immunofluorescence, Fluorescence

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