3031l  (Cell Signaling Technology Inc)

Journal: Immunity, Inflammation and Disease

Article Title: Transcription factor Krüppel‐like factor 4 upregulated G protein‐coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia–reperfusion injury

doi: 10.1002/iid3.940

Figure Lengend Snippet: GPR30 blocks OGD/R‐induced inflammation and apoptosis in Caco‐2 cells. Untransfected and transfected Caco‐2 cells were exposed to OGD/R, and the production of pro‐inflammatory cytokines, including (A) TNF‐α, (B) IL‐6, (C) IL‐1β, and (D) IL‐18 was assessed by ELISA. (E) The protein expression of p‐p65, p65, Cox‐2, and iNOS was detected using western blot. (F) TUNEL assay was applied to evaluate cell apoptosis. (G) The protein expression of Bcl‐2, Bax, cleaved‐PARP, and PARP was detected using western blot. ELISA, enzyme‐linked immunosorbent assay; OGD/R, oxygen‐glucose deprivation/reoxygenation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. *** p < .001 versus control; ## p < .01, ### p < .001 versus OGD/R+oe‐NC.

Article Snippet: After being blocked with skimmed milk, the membranes were probed with primary antibodies against GPR30 (ab39742, Abcam), p‐p65 (#3031, Cell Signaling Technology), p65 (#8242, Cell Signaling Technology), Cox2 (ab179800, Abcam), iNOS (ab178945, Abcam), Bcl‐2 (ab32124, Abcam), Bax (ab32503, Abcam), cleaved‐PARP (ab32064, Abcam), PARP (ab32138, Abcam), Claudin‐1 (ab211737, Abcam), Occludin‐1 (ab216327, Abcam), ZO‐1 (ab216880, Abcam), KLF4 (ab215036, Abcam), nod‐like receptor pyrin 3 (NLRP3; ab263899, Abcam), Cleaved‐caspase1 (#4199, Cell Signaling Technology), caspase1 (#3866, Cell Signaling Technology), N‐terminal Gasdermin D (GSDMD‐N; ab215203, Abcam), and GAPDH (ab9485, Abcam) at 4°C overnight, after which was the cultivation with horse radish peroxidase (HRP)‐conjugated secondary antibody (ab6721, Abcam) at room temperature for 2 h. The visualization and analysis of protein blots were operated employing enhanced chemiluminescence and Image LabTM software 5.2.1 (Bio‐Rad Laboratories, Inc), respectively.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, TUNEL Assay, Control

Journal: Immunity, Inflammation and Disease

Article Title: Transcription factor Krüppel‐like factor 4 upregulated G protein‐coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia–reperfusion injury

doi: 10.1002/iid3.940

Figure Lengend Snippet: KLF4 knockdown partially restrains the protective role of GPR30 against OGD/R‐induced inflammation and apoptosis in Caco‐2 cells. Caco‐2 cells were transfected with sh‐KLF4‐1 or sh‐KLF4‐2, and the (A) mRNA level and (B) protein expression of KLF4 were detected using RT‐qPCR and western blot, respectively. *** p < .001 versus sh‐NC. Then, Caco‐2 cells were transfected with oe‐GPR30 alone or cotransfected with oe‐GPR30 and sh‐NC/sh‐KLF4‐1, following OGD/R exposure. The production of pro‐inflammatory cytokines, including (C) TNF‐α, (D) IL‐6, (E) IL‐1β, and (F) IL‐18, was assessed by ELISA. (G) The protein expression of p‐p65, p65, Cox‐2, and iNOS was detected using western blot. (H, I) TUNEL assay was applied to evaluate cell apoptosis. (J) The protein expression of Bcl‐2, Bax, cleaved‐PARP, and PARP was detected using western blot. OGD/R, oxygen‐glucose deprivation/reoxygenation; RT‐qPCR, real‐time quantitative PCR. *** p < .001 versus control; ## p < .01, ### p < .001 versus OGD/R; @ p < .05, @@ p < .01, @@@ p < .001 versus OGD/R+oe‐GPR30+sh‐NC.

Article Snippet: After being blocked with skimmed milk, the membranes were probed with primary antibodies against GPR30 (ab39742, Abcam), p‐p65 (#3031, Cell Signaling Technology), p65 (#8242, Cell Signaling Technology), Cox2 (ab179800, Abcam), iNOS (ab178945, Abcam), Bcl‐2 (ab32124, Abcam), Bax (ab32503, Abcam), cleaved‐PARP (ab32064, Abcam), PARP (ab32138, Abcam), Claudin‐1 (ab211737, Abcam), Occludin‐1 (ab216327, Abcam), ZO‐1 (ab216880, Abcam), KLF4 (ab215036, Abcam), nod‐like receptor pyrin 3 (NLRP3; ab263899, Abcam), Cleaved‐caspase1 (#4199, Cell Signaling Technology), caspase1 (#3866, Cell Signaling Technology), N‐terminal Gasdermin D (GSDMD‐N; ab215203, Abcam), and GAPDH (ab9485, Abcam) at 4°C overnight, after which was the cultivation with horse radish peroxidase (HRP)‐conjugated secondary antibody (ab6721, Abcam) at room temperature for 2 h. The visualization and analysis of protein blots were operated employing enhanced chemiluminescence and Image LabTM software 5.2.1 (Bio‐Rad Laboratories, Inc), respectively.

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Real-time Polymerase Chain Reaction, Control

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: Treatment with CXCL5 neutralizing antibody upregulated VEGF/SDF-1 expression and promoted angiogenesis in late-EPCs from type 2 DM patients. Plasma levels of CXCL5 in type 2 DM patients and non-DM subjects (n = 6; A ). EPCs medium levels of CXCL5 in type 2 DM patients and non-DM subjects (n = 6; B ). The network formation and migration abilities were improved after the administration of CXCL5 mAb in EPCs from type 2 DM patients (n = 3; C , D ). Western blotting and statistical analyses of VEGF and SDF-1 in EPCs from type 2 DM patients (n = 3; E ). CXCL5 Chemokine C-X-C motif ligand 5, DM diabetes mellitus, EPC endothelial progenitor cell, mAb monoclonal antibody, SDF-1 stromal cell-derived factor 1, VEGF vascular endothelial growth factor. N represents cells cultured from n different individuals, and cells cultured from each individual were experimented for three independent experiments. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Expressing, Migration, Western Blot, Derivative Assay, Cell Culture, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: Treatment with CXCL5 neutralizing antibody upregulated VEGF/SDF-1 expression and promoted angiogenesis in late-EPCs from non-DM subjects and HAECs under the HG conditions. The network formation and migration abilities were improved after the administration of CXCL5 mAb in EPCs from non-DM subjects (n = 3; A , B ). Western blotting and statistical analyses of VEGF and SDF-1 in EPCs from non-DM subjects (n = 3; C ). The network formation and migration abilities were improved after the administration of CXCL5 mAb in HAECs (n = 3; D , E ). Western blotting and statistical analyses of VEGF and SDF-1 in HAECs (n = 3; F ). CXCL5 C-X-C motif chemokine ligand 5, EPC endothelial progenitor cell, HG high glucose, HAEC human aortic endothelial cell, mAb ,monoclonal antibody, SDF-1 stromal cell-derived factor 1, VEGF vascular endothelial growth factor. N represents the number of independent experiments on different days and in different experimental runs. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Expressing, Migration, Western Blot, Derivative Assay, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: CXCL5 impaired vascular endothelial function via the ERK/p65 signaling pathway in HAECs. The network formation and migration abilities were impaired after administration of CXCL5 for 2 days (n = 3; A , B ). Western blotting and statistical analyses of p-ERK, p-p65, IL-1β, IL-6, and TNF-α after administration of CXCL5 for 2 days (n = 3; C , D ). Western blotting and statistical analyses of VEGF and SDF-1 after administration of CXCL5 for 2 days (n = 3; E ). Western blotting of CXCR2 and CXCL5 after anti-goat IgG and CXCR2 immunoprecipitation (n = 3; F ). CXCL5 Chemokine C-X-C motif ligand 5, CXCR2 Chemokine C-X-C motif receptor 2, ERK extracellular signal-regulated kinase, HAEC human aortic endothelial cell, IL interleukin, SDF-1 stromal cell-derived factor 1, TNF-α tumor necrosis factor-α, VEGF vascular endothelial growth factor. N represents the number of independent experiments on different days and in different experimental runs. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Migration, Western Blot, Immunoprecipitation, Derivative Assay, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: CXCL5 neutralizing antibody repaired neovascularization and wound healing in type 1 DM mice. Serum CXCL5 levels in the diabetic mice were higher than those in the non-DM control. Treatment with CXCL5 mAb reduced CXCL5 levels (n = 6; A ). Representative evaluation of the ischemic (right) and nonischemic (left) hindlimbs before, immediately after 2 weeks and 4 weeks after the hindlimb ischemia surgery in STZ induced type 1 diabetic mice (n = 6; B ). The number of circulating EPCs was determined by flow cytometry in STZ induced type 1 diabetic mice. Treatment with CXCL5 mAb 100 μg increased the number of circulating EPCs after ischemia surgery compared with DM (n = 6; C ). Anti-CD31 immunostaining showed that CXCL5 mAb 100 μg treatment significantly increased the number of capillaries. Scale bar, 50 µm (n = 6; D ) Western blotting and statistical analyses of VEGF and SDF-1 in the ischemia leg (n = 3; E ). Angiogenesis in aortic ring cultures from CXCL5 mAb 100 μg mice was significantly increased the number of vessels sprouting compared with DM mice. Scale bar, 50 µm (n = 3; F ). Representative matrigel plug images and analysis of hemoglobin content (n = 6; G ). Representative matrigel plug images with immunostaining of CD31. CD31 positive areas were enhanced in the CXCL5 mAb 100 μg treatment mice. Scale bar, 50 µm ( H ). CXCL5 mAb 100 μg treatment improved wound repair ability in STZ induced type 1 diabetic mice. Representative wound areas and the closure rates of 3-mm punch biopsies were measured (n = 6; I ). Representative wound area images with immunostaining of CD31. CD31 positive areas were enhanced in the CXCL5 100 μg mAb treatment mice. Scale bar, 50 µm ( J ). Representative skin images with masson trichrome staining. Collagen depositions were enhanced in the CXCL5 mAb 100 μg treatment mice. Scale bar, 50 and 500 µm ( K ). CXCL5 Chemokine C-X-C motif ligand 5, DM diabetes mellitus, EPC endothelial progenitor cell, mAb monoclonal antibody, STZ Streptozotocin, SDF-1 stromal cell-derived factor 1, VEGF vascular endothelial growth factor. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01 compared with the non-DM control. # p < 0.05, ## p < 0.01 compared with the untreated DM group

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Flow Cytometry, Immunostaining, Western Blot, Staining, Derivative Assay, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: The neovascularization and wound healing were improved by neutralization of CXCL5 antibodies in type 2 DM mice. Treatment with CXCL5 mAb reduced CXCL5 levels in diabetic mice (n = 6; A ). Representative evaluation of the ischemic (right) and nonischemic (left) hindlimbs before, immediately after 2 weeks and 4 weeks after the hindlimb ischemia surgery in db/db mice (n = 6; B ). The number of circulating EPCs was determined by flow cytometry in db/db mice. Treatment with CXCL5 mAb 100 μg increased the number of circulating EPCs after ischemia surgery compared with DM (n = 6; C ). Anti-CD31 immunostaining showed that CXCL5 mAb 100 μg treatment significantly increased the number of capillaries. Scale bar, 50 µm (n = 6; D ). Western blotting and statistical analyses of VEGF and SDF-1 in the ischemia leg (n = 3; E ). Representative matrigel plug images and analysis of hemoglobin content (n = 6; F ). Representative matrigel plug images with immunostaining of CD31. CD31 positive areas were enhanced in the CXCL5 mAb 100 μg treatment mice. Scale bar, 50 µm ( G ). CXCL5 mAb 100 μg treatment improved wound repair ability in db/db mice. Representative wound areas and the closure rates of 3-mm punch biopsies were measured (n = 6; H ). Representative wound area images with immunostaining of CD31. CD31 positive areas were enhanced in the CXCL5 mAb 100 μg treatment mice. Scale bar, 50 µm ( I ). Serum concentration of VEGF and SDF-1 were higher in the CXCL5 mAb 100 μg treatment mice (n = 6; J , K ). CXCL5 Chemokine C-X-C motif ligand 5, DM diabetes mellitus, EPC endothelial progenitor cell, mAb monoclonal antibody, SDF-1 stromal cell-derived factor 1, VEGF vascular endothelial growth factor. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01 compared with the non-DM control. # p < 0.05, ## p < 0.01 compared with the untreated DM group

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Neutralization, Flow Cytometry, Immunostaining, Western Blot, Concentration Assay, Derivative Assay, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: The neovascularization and wound healing were improved in STZ-induced CXCL5KO diabetic mice. CXCL5 knockout mice had rarely circulating CXCL5 (n = 6; A ). Representative evaluation of the ischemic (right) and nonischemic (left) hindlimbs before, immediately after 2 weeks and 4 weeks after the hindlimb ischemia surgery in STZ induced type 1 diabetic mice (n = 6; B ). The number of circulating EPCs was determined by flow cytometry in STZ induced type 1 diabetic mice. Deletion of CXCL5 expression increased the number of circulating EPCs after ischemia surgery compared with DM (n = 6; C ). Anti-CD31 immunostaining showed that inhibition of CXCL5 expression significantly increased the number of capillaries. Scale bar, 50 µm (n = 6; D ). Western blotting and statistical analyses of VEGF and SDF-1 in the ischemia leg (n = 3; E ). Representative matrigel plug and analysis of hemoglobin content (n = 6; F ). Representative matrigel plug images with immunostaining of CD31. CD31 positive areas were enhanced in the CXCL5 knockout diabetic mice. Scale bar, 50 µm ( G ). CXCL5 inhibition by knockout improved wound repair ability in STZ induced type 1 diabetic mice. Representative wound areas and the closure rates of 3-mm punch biopsies were measured (n = 6; H ). Representative wound area images with immunostaining of CD31. CD31 positive areas were enhanced in the CXCL5 knockout mice. Scale bar, 50 µm ( I ). CXCL5, Chemokine C-X-C motif ligand 5; CXCL5KO, CXCL5-knockout mice; CXCL5KO+STZ, CXCL5 knockout diabetic mice. DM diabetes mellitus, EPC endothelial progenitor cell, STZ streptozotocin, WT wild-type mice, WT+STZ wild-type diabetic mice. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01 compared with the WT group. # p < 0.05, ## p < 0.01 compared with the WT+STZ group

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Knock-Out, Flow Cytometry, Expressing, Immunostaining, Inhibition, Western Blot, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: Summary of beneficial effects of CXCL5 suppression in diabetic vasculopathy. CXCL5 Chemokine C-X-C motif ligand 5, CXCR2 Chemokine C-X-C motif receptor 2, EPC endothelial progenitor cell, ERK extracellular signal-regulated kinase, DM diabetes mellitus, IL interleukin, SDF-1 stromal cell-derived factor 1, TNF-α tumor necrosis factor-α, VEGF vascular endothelial growth factor

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

doi: 10.1186/s12933-023-01900-w

Figure Lengend Snippet: CXCL5 impaired vascular endothelial function via the ERK/p65 signaling pathway in HAECs. The network formation and migration abilities were impaired after administration of CXCL5 for 2 days (n = 3; A , B ). Western blotting and statistical analyses of p-ERK, p-p65, IL-1β, IL-6, and TNF-α after administration of CXCL5 for 2 days (n = 3; C , D ). Western blotting and statistical analyses of VEGF and SDF-1 after administration of CXCL5 for 2 days (n = 3; E ). Western blotting of CXCR2 and CXCL5 after anti-goat IgG and CXCR2 immunoprecipitation (n = 3; F ). CXCL5 Chemokine C-X-C motif ligand 5, CXCR2 Chemokine C-X-C motif receptor 2, ERK extracellular signal-regulated kinase, HAEC human aortic endothelial cell, IL interleukin, SDF-1 stromal cell-derived factor 1, TNF-α tumor necrosis factor-α, VEGF vascular endothelial growth factor. N represents the number of independent experiments on different days and in different experimental runs. The Mann–Whitney U test was used to determine statistically significant differences. *p < 0.05, **p < 0.01

Article Snippet: After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight.

Techniques: Migration, Western Blot, Immunoprecipitation, Derivative Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Fermented Sprouts of Codonopsis lanceolata Suppress LPS-Induced Inflammatory Responses by Inhibiting NF-κB Signaling Pathway in RAW 264.7 Macrophages and CD1 Mice

doi: 10.3390/pharmaceutics15071793

Figure Lengend Snippet: Attenuation of MAPK phosphorylation and NF-κB signaling by F-CSA. ( A ) The expression of phosphorylated p65 (p-p65), ERK (p-ERK), p38 (p-p38), JNK (p-JNK), p65, ERK, p38, JNK. The phosphorylation level of ( B ) p65 and ( C ) JNK was normalized to control. ### p < 0.001 compared to vehicle; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to LPS alone.

Article Snippet: Antibodies against p38, JNK, ERK, p65, phosphorylated p38 (p-p38), phosphorylated JNK (p-JNK), phosphorylated ERK (p-ERK), phosphorylated p65 (p-p65), iNOS, COX-2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Expressing, Control

Journal: Chinese Medicine

Article Title: Mechanisms of glabridin inhibition of integrin α IIb β 3 inside-out signals and NF-κB activation in human platelets

doi: 10.1186/s13020-023-00779-9

Figure Lengend Snippet: Inhibitory activity of glabridin, BAY11-7082, and Ro106-9920 on the activation of NF-κB in human platelets. Washed platelets were preincubated with 0.1% DMSO, glabridin (30 µM), BAY11-7082 (10 µM), or Ro106-9920 (10 µM), followed by the addition of collagen (1 µg/mL) to trigger NF-κB activation for the immunoblotting of A IKK, B IκBα, and C p65 phosphorylation as well as D IκBα degradation. Data are presented as mean ± standard error of the mean ( n = 4). *** P < 0.001, compared with the resting control (Tyrode’s solution); ### P < 0.001, compared with the 0.1% DMSO-treated group

Article Snippet: Anti-phospho-(Ser) PKC substrate, anti-IκBα (44D4), and anti-phospho-NF-κB p65 (Ser 536 ) pAbs, anti-phospho-IκBα (Ser 32/36 ; 5A5), and phospho-IKKα/β (Ser 176/180 ; 16A6) monoclonal antibodies (mAbs) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Activity Assay, Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: Chinese Medicine

Article Title: Mechanisms of glabridin inhibition of integrin α IIb β 3 inside-out signals and NF-κB activation in human platelets

doi: 10.1186/s13020-023-00779-9

Figure Lengend Snippet: Effects of glabridin, BAY11-7082, and Ro106-9920 in regulating collagen-stimulated NF-κB activation measured using confocal laser fluorescence. Washed platelets were preincubated with 0.1% DMSO, glabridin (30 µM), BAY11-7082 (10 µM), or Ro106-9920 (10 µM), followed by the addition of collagen (1 µg/mL) to trigger platelet activation. The confocal images of A , B IKK (green fluorescence), C , D p-p65 (green fluorescence), and α-tubulin (blue fluorescence) were observed using goat anti-rabbit CF TM 488A and goat anti-mouse CF TM 405M dyes, respectively, as described in “ ”. The confocal images represent four similar experiments. bar: 2.5 μm. The intensity of green fluorescence representing the A , B phospho-IKK and C , D phospho-p65 were quantified in at least four different fields per view (mean fluorescence intensity [MFI]). Data are presented as mean ± standard error of the mean. *** P < 0.001, compared with the resting control (Tyrode’s solution); ### P < 0.001, compared with the 0.1% DMSO-treated group

Article Snippet: Anti-phospho-(Ser) PKC substrate, anti-IκBα (44D4), and anti-phospho-NF-κB p65 (Ser 536 ) pAbs, anti-phospho-IκBα (Ser 32/36 ; 5A5), and phospho-IKKα/β (Ser 176/180 ; 16A6) monoclonal antibodies (mAbs) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Activation Assay, Fluorescence, Control