Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS‐primed MSCs into wounds increased the expression of CXCL6, IL‐8, and IL‐1β. Graphs display numbers of double‐positive cells for h‐β2M + CXCL6, h‐β2M + IL‐8, and h‐β2M + IL‐1β, respectively. MSC‐injected wounds served as controls. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50 μm. Results show that LPS‐primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL‐8 in mice and is known as neutrophil chemoattractant. Similar results were found for IL‐1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP‐2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double‐positive cells for Ly6G MIP/KC, IL‐1β, and LIX, respectively. PBS and non‐primed MSC‐injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Scale bars: 50 μm.

Article Snippet: In addition, we employed primary antibodies against α‐SMA (Progen), CD206 (Biorbyt), CXCL5 (Gene Tex), CXCL6 (Biorbyt), IL‐1β (Abcam), IL‐6 (R&D), IL‐8 (R&D), β2M (Signaling), F4/80 (eBioscience), TGFβ‐1 and CD31 (Cell Signaling), MIP2/KC and GCSF (R&D), and LIX (Biorbyt).

Techniques: Injection, Expressing, Comparison, Functional Assay, Sequencing

Journal: World Journal of Gastroenterology

Article Title: Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

doi: 10.3748/wjg.v23.i28.5167

Figure Lengend Snippet: Expression levels of stromal cell-derived factor-1, CXC chemokine receptor 4 and granulocyte chemotactic protein-2 in colon cancer cell lines and stromal cells. The protein expression levels of CXCL2, CXCR4 and CXCL6 in colon cancer cell lines and stromal cells were determined in whole-cell lysates by western blotting analysis. Thirty micrograms of total cell lysate were subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with antibodies to CXCL12, CXCR4 and CXCL6. β-actin was used as a loading control. CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; CXCR4: CXC chemokine receptor 4.

Article Snippet: Recombinant human CXCL6 and CXCL12 were purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Expressing, Derivative Assay, Western Blot, SDS Page, Membrane, Control

Journal: World Journal of Gastroenterology

Article Title: Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

doi: 10.3748/wjg.v23.i28.5167

Figure Lengend Snippet: Enhancement of secreted granulocyte chemotactic protein-2 levels in colon cancer cell lines and stromal cells by recombinant stromal cell-derived factor-1 and co-culture with fibroblasts. The alteration of CXCL6 secretion from colon cancer cell lines [CaCo-2 (A), WiDr (B), HT-29 (C) and DLD-1 (D)] by recombinant CXCL12 stimulation or co-culture with fibroblasts (FB) were determined by enzyme-linked immunosorbent assay in cell culture medium. Meanwhile, colon cancer cells were treated with anti-CXCL12 antibody (Ab) for 2 h, and the concentration of CXCL6 was measured by ELISA in supernatants from colon cancer cells. Effect on secretion of CXCL6 from HUVECs stimulated by recombinant CXCL12 in co-culture system with fibroblasts and the colon cancer cells DLD-1 are shown (E). The experimental detail is described in the “Materials and Methods” section. Control: colon cancer cells only; FB: fibroblasts only; CXCL12: treated with recombinant CXCL12; with FB: colon cancer cells co-cultured with fibroblasts; with FB + Ab: colon cancer cells co-cultured with fibroblasts and pre-treated with anti-CXCL12 Ab. The values are expressed as mean ± SD. Ab: Antibody; CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.

Article Snippet: Recombinant human CXCL6 and CXCL12 were purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Recombinant, Derivative Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Control

Journal: World Journal of Gastroenterology

Article Title: Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

doi: 10.3748/wjg.v23.i28.5167

Figure Lengend Snippet: Effect of stromal cell-derived factor-1, granulocyte chemotactic protein-2 and conditioned medium from fibroblasts on human umbilical vein endothelial cell proliferation. HUVECs were cultured in medium containing different concentrations of CXCL6 (A), CXCL12 (B) and conditioned medium from fibroblasts. After 72 h of incubation, HUVEC proliferation was assessed using premixed WST-1 cell proliferation assay (column mean absorbance reading; Bars = SD). Multiple comparisons were performed by one-way ANOVA followed by the SNK test; a P < 0.05, b P < 0.01. CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.

Article Snippet: Recombinant human CXCL6 and CXCL12 were purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Derivative Assay, Cell Culture, Incubation, Proliferation Assay

Journal: World Journal of Gastroenterology

Article Title: Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

doi: 10.3748/wjg.v23.i28.5167

Figure Lengend Snippet: Effect of granulocyte chemotactic protein-2, stromal cell-derived factor-1 and co-culture with fibroblasts or DLD-1 on colon cancer cell and human umbilical vein endothelial cell invasiveness. The influence of different concentrations of CXCL6 (A), CXCL12 (B) or co-culture with fibroblasts on colon cancer cell invasiveness was measured by the BD Bio-Coat Matrigel invasion assay system (BD Biosciences). HT-29 (A and B) cells and HUVECs (C and D) were pre-treated with different concentrations of CXCL6 and CXCL12, and co-culture with fibroblasts (E) or DLD-1 (F), or pre-treated with or without anti CXCL6 or CXCL12 antibody, and following a 24-h incubation. The invading cells were fixed and stained with Diff-Quick stain. Invading cells were counted in five random microscopic fields (× 200). The experiment detail is described in the “Material and Methods” section. Multiple comparisons were performed by one-way ANOVA followed by the SNK test; a P < 0.05, b P < 0.01. A1: HT-29 cells only; A2: 0.1 ng/mL of CXCL6; A3: 1 ng/mL of CXCL6; A4: 10 ng/mL of CXCL6. B1: HT-29 cells only; B2: 0.1 ng/mL of CXCL12; B3: 1 ng/mL of CXCL12; B4: 10 ng/mL of CXCL12; C1: HUVECs only; C2: 1 ng/mL of CXCL6; C3: 10 ng/mL of CXCL6; C4: 10 ng/mL of CXCL6 treated with 10 μg/mL CXCL6 Ab. D1: HUVECs only; D2: 1 ng/mL of CXCL12; D3: 10 ng/mL of CXCL12; D4: 10 ng/mL of CXCL12 treated with 10 μg/mL CXCL12 Ab. E1: HUVECs only; E2: HUVECs co-culture with fibroblasts; E3: Co-culture with fibroblasts + 10 μg/mL CXCL12 Ab; E4: Co-culture with fibroblasts + 10 ng/mL CXCL6. F1: HUVECs only; F2: HUVECs co-culture with DLD-1 cells; F3: Co-culture with DLD-1 cells + 10 μg/mL CXCL12 Ab; F4: Co-culture with CaCo-2 cells. Ab: Antibody; CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.

Article Snippet: Recombinant human CXCL6 and CXCL12 were purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Derivative Assay, Co-Culture Assay, Invasion Assay, Incubation, Staining, Diff-Quik

Journal: World Journal of Gastroenterology

Article Title: Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

doi: 10.3748/wjg.v23.i28.5167

Figure Lengend Snippet: Effect of granulocyte chemotactic protein-2, stromal cell-derived factor-1 and co-culture with colon cancer cells on angiogenesis. The treatment of CXCL6 (A) and CXCL12 (B) influence HUVEC tube formation. After incubation of the HUVEC/fibroblast co-culture system in the presence or absence of CXCL6 or anti-CXCL12 Ab, then co-culture for 7 d, the HUVEC/fibroblast co-culture system was stained with anti-CD31 antibody. Tube formation area was measured quantitatively using an image analyzer. A1: Control; A2: 1 ng/mL CXCL6; A3: 10 ng/mL CXCL6; A4: 10 ng/mL CXCL6 + 10 μg/mL of CXCL6 Ab. B1: Control; B2: 1 ng/mL CXCL12; B3: 10 ng/mL CXCL12; B4: 10 ng/mL CXCL12 + 10 μg/mL of CXCL12 Ab. Effect of colon cancer cells (DLD-1, HT-29 or CaCo-2) on HUVEC tube formation is shown (C). Angiogenesis assay by HUVEC/fibroblast co-culture with DLD-1, HT-29 or CaCo-2 cells was conducted using the double-chamber method. Detection of tube formation by HUVECs was described in the “Material and Methods” section. C1: Co-culture with DLD-1; C2: Co-culture with DLD-1 + 10 ng/mL of CXCL6; C3: Co-culture with DLD-1 + 10 ng/mL of CXCL12; C4: Co-culture with DLD-1 + 10 μg/mL of CXCL12 Ab; C5: Co-culture with HT-29 cells; C6: Co-culture with HT-29 cells pre-treated with 10 ng/mL CXCL6; C7: Co-culture with HT-29 cells pre-treated with 10 ng/mL CXCL12; C8: Co-culture with HT-29 + 10 μg/mL of CXCL12 Ab; C9: Co-culture with CaCo-2 cells; C10: Co-culture with CaCo-2 cells pre-treated with 10 ng/mL CXCL6; C11: Co-culture with CaCo-2 cells pre-treated with 10 ng/mL CXCL12; C12: Co-culture with CaCo-2 cells pre-treated with 10 μg/mL anti-CXCL12 antibody. Columns, mean pixels of HUVEC tube formation area; Bars = SD. Multiple comparisons were performed by one-way ANOVA followed by the SNK test; b P < 0.01 vs control. Ab: Antibody; CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.

Article Snippet: Recombinant human CXCL6 and CXCL12 were purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Derivative Assay, Co-Culture Assay, Incubation, Staining, Control, Angiogenesis Assay

Journal: World Journal of Gastroenterology

Article Title: Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

doi: 10.3748/wjg.v23.i28.5167

Figure Lengend Snippet: Stromal cell-derived factor-1-induced phosphorylation of PI3K/Akt/mTOR signaling in colon cancer cell lines and stromal cells. HT-29 cells and HUVECs were treated with 10 ng/mL of CXCL12 cultured for 5, 10 and 30 min. The cells were collected and lysed by lysis buffer. Aliquots of 30 μg of lysed protein were subjected to immunoblotting with a phospho-Akt (A), phospho-PI3K (B) and phosphor-mTOR (C) Abs. Detection of total Akt, PI3K or mTOR levels aided in loading control. HT-29 cells or HUVECs, after being pre-treated with 50 μmol/L Akt inhibitor and 50 μmol/L LY294002 for 1 h, were incubated with 10 ng/mL CXCL12 for 1 h. Results of immunoblotting using the mTOR Ab is shown (D). Detection of total mTOR levels served as loading control. Ab: Antibody; CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.

Article Snippet: Recombinant human CXCL6 and CXCL12 were purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Derivative Assay, Phospho-proteomics, Cell Culture, Lysis, Western Blot, Control, Incubation

Journal: Cancer Cell International

Article Title: Ring finger protein 152-dependent degradation of TSPAN12 suppresses hepatocellular carcinoma progression

doi: 10.1186/s12935-021-01806-1

Figure Lengend Snippet: RNF152 governed HCC progression partially dependent on TSPAN12 degradation. a HuH6 cells were transfected with or without shRNA against RNF152 individually or simultaneously with TSPAN12. The cell lysates were detected by immunoblotting with indicated antibodies. b HuH6 cells in a were subjected to BrdU test. ***P < 0.001, **P < 0.01. c HuH6 cells in a were examined for colony formation. **P < 0.01. d HuH6 cells in a were examined for cell invasion. **P < 0.01. e CXCL6 mRNA expression was regulated by TSPAN12. The mRNA levels of CXCL6 in a were determined by real-time PCR. **P < 0.01. f The production of CXCL6 secreted from cells in a was quantified by ELISA. **P < 0.01, ***P < 0.001. g Each nude mouse was subcutaneously injected with 1 × 10 7 HuH6 cells in a , and continued observation for 4 weeks. Tumour growth was measured using a caliper at the indicated times after injection. n = 4 for each group. ***P < 0.001. h Tumor weights were measured after mice were sacrificed. **P < 0.01, *P < 0.05

Article Snippet: The antigen–antibody reaction was performed using DuoSet ELISA for human CXCL6 (R&D Systems) according to the manufacturer’s instructions.

Techniques: Transfection, shRNA, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Injection

Journal: Frontiers in Medicine

Article Title: miR-6869-5p Transported by Plasma Extracellular Vesicles Mediates Renal Tubule Injury and Renin-Angiotensin System Activation in Obesity

doi: 10.3389/fmed.2021.725598

Figure Lengend Snippet: The list of primers sequences.

Article Snippet: We used the following primary antibodies against several EV-characteristic markers: CD9 (#ab92726, Abcam, Cambridge, MA, USA), CD81 (#sc-7637, 1:200, Santa Cruz Biotechnology, USA), CD63 (#ab59479, Abcam, 1:1,000), AGT (AF3156, R&D Systems, Minneapolis, MN, 1:1,000), ACE2 (AF333, R&D Systems, 1:1,000), ACE (AF929, R&D Systems, 1:1,000), AT1 (MAB102441, R&D Systems, 1:1,000), KIM-1 (NBP1-76701SS, Novus, 1:1,000), and NGAL (AF1757-SP, R&D Systems, 1:1,000).

Techniques:

Journal: Frontiers in Medicine

Article Title: miR-6869-5p Transported by Plasma Extracellular Vesicles Mediates Renal Tubule Injury and Renin-Angiotensin System Activation in Obesity

doi: 10.3389/fmed.2021.725598

Figure Lengend Snippet: Effects of plasma EVs on renal tubule injury and RAS activation in obesity. (A–C) After treating PTECs with PBS(negative control), Lean-EVs, or Obese-EVs, the relative mRNA and protein levels of AGT, ACE, ACE2, and AT1 were analyzed by qRT-PCR (A) and Western blotting (B,C) ( n = 3 per group). (D,E) Levels of KIM1 and NGAL protein in PTECs treated with PBS, Lean-EVs, or Obese-EVs. Data are presented as mean ± SD; ** P < 0.001 vs. PBS, * P < 0.001 vs. Lean-EVs. EVs, extracellular vesicles; PTECs, Proximal tubular epithelial cells; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; AGT, angiotensinogen; ACE, angiotensin-converting enzyme; ACE2, angiotensin-converting enzyme 2; AT1, angiotensin 1.

Article Snippet: We used the following primary antibodies against several EV-characteristic markers: CD9 (#ab92726, Abcam, Cambridge, MA, USA), CD81 (#sc-7637, 1:200, Santa Cruz Biotechnology, USA), CD63 (#ab59479, Abcam, 1:1,000), AGT (AF3156, R&D Systems, Minneapolis, MN, 1:1,000), ACE2 (AF333, R&D Systems, 1:1,000), ACE (AF929, R&D Systems, 1:1,000), AT1 (MAB102441, R&D Systems, 1:1,000), KIM-1 (NBP1-76701SS, Novus, 1:1,000), and NGAL (AF1757-SP, R&D Systems, 1:1,000).

Techniques: Clinical Proteomics, Activation Assay, Negative Control, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Medicine

Article Title: miR-6869-5p Transported by Plasma Extracellular Vesicles Mediates Renal Tubule Injury and Renin-Angiotensin System Activation in Obesity

doi: 10.3389/fmed.2021.725598

Figure Lengend Snippet: Plasma Obese-EVs induce renal tubule injury and RAS activation in PTECs via transport of miR-6869-5p. (A–D) After in vitro transfection of miR-6869-5p mimic and miR-NC-mimic, the protein levels of AGT, ACE, ACE2, AT1 (A,B) , KIM-I, and NAGAL (C,D) were analyzed by Western blotting ( n = 3). (E,F) After transfection with miR-6869-5p inhibitor or miR-NC-inhibitor for 48 h, PTECs were treated with PBS, Lean-EVs, or Obese-EVs. The protein levels of AGT, ACE, ACE2, AT1 (E,F) , KIM-I, and NGAL (G,H) were analyzed by Western blotting ( n = 3). Data are presented as mean ± SD; ** P <0.001 vs. PBS, * P <0.001 vs. Lean-EVs. EVs, extracellular vesicles; PTECs, Proximal tubular epithelial cells; AGT, angiotensinogen; ACE, angiotensin-converting enzyme; ACE2, angiotensin-converting enzyme 2; AT1, angiotensin 1; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin.

Article Snippet: We used the following primary antibodies against several EV-characteristic markers: CD9 (#ab92726, Abcam, Cambridge, MA, USA), CD81 (#sc-7637, 1:200, Santa Cruz Biotechnology, USA), CD63 (#ab59479, Abcam, 1:1,000), AGT (AF3156, R&D Systems, Minneapolis, MN, 1:1,000), ACE2 (AF333, R&D Systems, 1:1,000), ACE (AF929, R&D Systems, 1:1,000), AT1 (MAB102441, R&D Systems, 1:1,000), KIM-1 (NBP1-76701SS, Novus, 1:1,000), and NGAL (AF1757-SP, R&D Systems, 1:1,000).

Techniques: Clinical Proteomics, Activation Assay, In Vitro, Transfection, Western Blot

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet: Schematic representation of IL-17/CXCL5 signaling in chronically injured cerebral endothelia (A). TRAP-qPCR fold expression compared with average fpkm of top DEGs from white matter endothelia (*adjusted p < 0.05) (B). Weight-adjusted ELISA values (pg/mL) for murine CXCL5 in retro-orbital blood samples from CFD (black) and HFD (red) animals (n = 4/grp, p = 0.0355) (C). Immunofluorescence labeling for IL-17Rb (green, D) and CXCL5 (green, E) is absent in white matter vasculature of Tie2-Cre;tdTomato mice on CFD (left panels) and abundant in white matter vasculature of Tie2-Cre;tdTomato mice on HFD (right panels). Single-channel labeling for IL17Rb (bottom panels, D) and CXCL5 (bottom panels, E) show heterogeneous endothelial expression. Labeling for GLUT-1 (blue), CXCL5 (red), and PDGFRα (green) at 7 days post-stroke in animals on CFD (left) and HFD (right). Inset boxes from the peri-infarct tissue (top) masked for GLUT-1 (white) with only co-localized CXCL5 (purple) (bottom). Graph of percentage of co-localized CXCL5+/GLUT-1+ voxels (****p < 0.0001) (F). Error bars represent S.E.M. Scale bars: 50 μm (F), 20 μm (D), and 10 μm (E).

Article Snippet: murine CXCL5 , Origene , #MR200761.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet: Human brain microvascular endothelial cells were stimulated with IL-17 ligands A–E (250 ng/mL) and CXCL5 levels measured in conditioned media 48 h after stimulation (*p = 0.0372 by Kruskal-Wallis H test; **post-hoc comparison for IL-17B versus no ligand, adjusted p = 0.0178) (A). Phalloidin+ cellular area in O4+ OPCs grown in vitro exposed to vehicle (top panel) or recombinant CXCL5 (bottom panel) for 48 h (p < 0.0001, F = 9.82 by one-way ANOVA) (B). Approach for CXCL5 transgenic-viral gain of function in subcortical white matter of Tie2-Cre;tdTomato mice (top panel) (C). PDGFRα+ OPC (green) labeling in GFP-transduced Tie2-Cre;tdTomato mice (red, left panel) and CXCL5-GFP-transduced Tie2-Cre;tdTomato mice (right panel). Representative masked cellular profiles of PDGFRα+ cell area (bottom panels). Schematic of anti-IL-17B antibody treatment (top panel) (D). PDGFRα+ OPC (green) labeling in control IgG-treated Tie2-Cre:tdT mice (left panel) and anti-IL-17B IgG-treated Tie2-Cre:tdT mice (right panel). Representative masked cellular profiles of PDGFRα+ cell area (bottom panels). Proportion of OPCs per unit distance from vessel (0–35 μm) in each condition (total measured cell number per condition in parentheses) (E). Average distance of OPCs to vessel (***p = 0.0005, F = 6.06 by one-way ANOVA; **adjusted p = 0.0039; *adjusted p = 0.0168) (F). Average in vivo PDGFRα+ OPC cell area (**p = 0.0068, F = 7.38 by one-way ANOVA; **adjusted p = 0.002) (G). Graph of co-localized CXCL5+/GLUT-1+ voxels in anti-IL-17B IgG-treated animals (n = 4/grp; *p = 0.018) (H). Error bars represent S.E.M. Scale bars: 10 μm

Article Snippet: murine CXCL5 , Origene , #MR200761.

Techniques: Comparison, In Vitro, Recombinant, Transgenic Assay, Labeling, Control, In Vivo

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet: Plasma levels of log 10 -CXCL5 in ASPIRE cohort subjects separated by detectable plasma IL-17B (n = 32; median 1043.0 pg/mL) compared with those with undetectable plasma IL-17B (n = 99; median 515.3 pg/mL; *p < 0.0001). Plasma log 10 -CXCL5 levels in subjects with MRI-confirmed acute microvascular ischemia (IL-17B + subjects; n = 9; 978.2 pg/mL versus IL-17B− subjects; n = 24; 539.7 pg/mL) (**p = 0.0157) (A). Ordinal shift analysis of modified Fazekas scale scores from plasma IL-17B+ and IL-17B− subjects (p < 0.0001) (B). Representative immunohistochemical detection of CXCL5 in human frontal white matter vasculature in subjects with cerebrovascular pathology (C). Percentage of CXCL5+ vessel segments in peri-ventricular white matter (n = 10) (p = 0.0005). Error bars represent S.E.M. Scale bar: 10 μm

Article Snippet: murine CXCL5 , Origene , #MR200761.

Techniques: Clinical Proteomics, Modification, Immunohistochemical staining

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet:

Article Snippet: murine CXCL5 , Origene , #MR200761.

Techniques: Blocking Assay, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Luminex, Hybridization, Plasmid Preparation, Biomarker Discovery, Control, Software

Journal: Nature Communications

Article Title: IL-17A is increased in diabetic wounds and impairs keratinocyte function via histone demethylase JMJD3

doi: 10.1038/s41467-025-67456-3

Figure Lengend Snippet: a ChIP analysis of H3K27me3 or IgG at indicated promoters in keratinocytes with (white) or without (blue) IL-17A stimulation. Itga3: n = 5 (unstimulated), n = 4 (IL-17A-stimulated) technical replicates, p = 0.0034, Timp1 : n = 3 technical replicates, p = 0.0046, Ccl20 : n = 3 technical replicates, p = 0.0059, Cxcl1 : n = 3 technical replicates, p = 0.0020, Cxcl3 : n = 3 technical replicates, p = 0.0258, Cxcl5 : n = 3 technical replicates, p = 0.0174. n = 3 independent experiments. b qPCR analysis of keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (1 µM) (red). Itga3 : n = 4 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0017 (IL-17A vs. IL-17A and inhibitor), Timp1: n = 6 biological replicates, p < 0.0312 (DMSO vs. IL-17A), p = 0.0029 (IL-17A vs. IL-17A and inhibitor), Ccl20: n = 3 biological replicates, p = 0.0008 (DMSO vs. IL-17A), p = 0.0428 (IL-17A vs. IL-17A and inhibitor), Cxcl1 : n = 3 biological replicates, p = 0.0003 (DMSO vs. IL-17A), p = 0.0294 (IL-17A vs. IL-17A and inhibitor), Cxcl3 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0003 (IL-17A vs. IL-17A and inhibitor), Cxcl5 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0007 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. c Western blot of ITGA-3 expression in keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). Representative densitometry plot is shown. n = 3 independent experiments. d Protein quantification of lysates from keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). TIMP-1: n = 6 biological replicates, p = 0.0012 (DMSO vs. IL-17A), p = 0.0068 (IL-17A vs. IL-17A and inhibitor), CCL-20: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0199 (IL-17A vs. IL-17A and inhibitor), CXCL-1: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0158 (IL-17A vs. IL-17A and inhibitor), CXCL-3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0167 (IL-17A vs. IL-17A and inhibitor), CXCL-5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0005 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. e qPCR analysis of keratinocytes treated with a non-targeting control (siNTC) (white) or si Jmjd3 (gray). Jmjd3 : n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0192, Itga3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Timp1: n = 3 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0205, Ccl20: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0015, Cxcl1: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Cxcl3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, Cxcl5: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, n = 3 independent experiments. f qPCR analysis of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (yellow) keratinocytes. n = 3 biological replicates, Itga3 : p = 0.0184, Timp1 : p = 0.0044, Ccl20 : p = 0.0160, Cxcl1 : p = 0.0371, Cxcl3 : p = 0.0042. n = 3 independent experiments. g , h Scratch assays of primary murine ( n = 3 biological replicates, p < 0.0001 (48 h)) and N/TERT ( n = 3 biological replicates, p = 0.0340 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J4 (red). n = 3 independent experiments. i , j Scratch assays of primary murine ( n = 3 biological replicates, p = 0.0016 (48 h)) and N/TERT ( n = 6 biological replicates (IL-17A alone), n = 4 biological replicates (IL-17A and GSK-J1), p = 0.0223 (8 h), p < 0.0001 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J1 (red). n = 3 independent experiments. k Scratch assay of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (blue) keratinocytes. n = 3 biological replicates, p = 0.0198 (48 h). n = 3 independent experiments. Data were analyzed for variances, and 2-tailed Student’s t tests for ( a ), ( e ), ( f ) and 1-way ANOVA tests for ( b ), ( d ), ( g – k ) were performed. Data are presented as the mean ± SEM.

Article Snippet: After stimulation, cell free supernatant was collected and analyzed by the University of Michigan Immune Monitoring Shared Resource Core for CCL-20, CXCL-1, CXCL-5 or specific enzyme immunoassay kits for CXCL-3 (Boster Bio) and TIMP-1 (R&D Systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Wound Healing Assay

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet: Schematic representation of IL-17/CXCL5 signaling in chronically injured cerebral endothelia (A). TRAP-qPCR fold expression compared with average fpkm of top DEGs from white matter endothelia (*adjusted p < 0.05) (B). Weight-adjusted ELISA values (pg/mL) for murine CXCL5 in retro-orbital blood samples from CFD (black) and HFD (red) animals (n = 4/grp, p = 0.0355) (C). Immunofluorescence labeling for IL-17Rb (green, D) and CXCL5 (green, E) is absent in white matter vasculature of Tie2-Cre;tdTomato mice on CFD (left panels) and abundant in white matter vasculature of Tie2-Cre;tdTomato mice on HFD (right panels). Single-channel labeling for IL17Rb (bottom panels, D) and CXCL5 (bottom panels, E) show heterogeneous endothelial expression. Labeling for GLUT-1 (blue), CXCL5 (red), and PDGFRα (green) at 7 days post-stroke in animals on CFD (left) and HFD (right). Inset boxes from the peri-infarct tissue (top) masked for GLUT-1 (white) with only co-localized CXCL5 (purple) (bottom). Graph of percentage of co-localized CXCL5+/GLUT-1+ voxels (****p < 0.0001) (F). Error bars represent S.E.M. Scale bars: 50 μm (F), 20 μm (D), and 10 μm (E).

Article Snippet: A plasmid containing the open reading frame of the murine CXCL5 sequence with a 3′ stop codon was purchased from Origene (#MR200761).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet: Human brain microvascular endothelial cells were stimulated with IL-17 ligands A–E (250 ng/mL) and CXCL5 levels measured in conditioned media 48 h after stimulation (*p = 0.0372 by Kruskal-Wallis H test; **post-hoc comparison for IL-17B versus no ligand, adjusted p = 0.0178) (A). Phalloidin+ cellular area in O4+ OPCs grown in vitro exposed to vehicle (top panel) or recombinant CXCL5 (bottom panel) for 48 h (p < 0.0001, F = 9.82 by one-way ANOVA) (B). Approach for CXCL5 transgenic-viral gain of function in subcortical white matter of Tie2-Cre;tdTomato mice (top panel) (C). PDGFRα+ OPC (green) labeling in GFP-transduced Tie2-Cre;tdTomato mice (red, left panel) and CXCL5-GFP-transduced Tie2-Cre;tdTomato mice (right panel). Representative masked cellular profiles of PDGFRα+ cell area (bottom panels). Schematic of anti-IL-17B antibody treatment (top panel) (D). PDGFRα+ OPC (green) labeling in control IgG-treated Tie2-Cre:tdT mice (left panel) and anti-IL-17B IgG-treated Tie2-Cre:tdT mice (right panel). Representative masked cellular profiles of PDGFRα+ cell area (bottom panels). Proportion of OPCs per unit distance from vessel (0–35 μm) in each condition (total measured cell number per condition in parentheses) (E). Average distance of OPCs to vessel (***p = 0.0005, F = 6.06 by one-way ANOVA; **adjusted p = 0.0039; *adjusted p = 0.0168) (F). Average in vivo PDGFRα+ OPC cell area (**p = 0.0068, F = 7.38 by one-way ANOVA; **adjusted p = 0.002) (G). Graph of co-localized CXCL5+/GLUT-1+ voxels in anti-IL-17B IgG-treated animals (n = 4/grp; *p = 0.018) (H). Error bars represent S.E.M. Scale bars: 10 μm

Article Snippet: A plasmid containing the open reading frame of the murine CXCL5 sequence with a 3′ stop codon was purchased from Origene (#MR200761).

Techniques: Comparison, In Vitro, Recombinant, Transgenic Assay, Labeling, Control, In Vivo

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet: Plasma levels of log 10 -CXCL5 in ASPIRE cohort subjects separated by detectable plasma IL-17B (n = 32; median 1043.0 pg/mL) compared with those with undetectable plasma IL-17B (n = 99; median 515.3 pg/mL; *p < 0.0001). Plasma log 10 -CXCL5 levels in subjects with MRI-confirmed acute microvascular ischemia (IL-17B + subjects; n = 9; 978.2 pg/mL versus IL-17B− subjects; n = 24; 539.7 pg/mL) (**p = 0.0157) (A). Ordinal shift analysis of modified Fazekas scale scores from plasma IL-17B+ and IL-17B− subjects (p < 0.0001) (B). Representative immunohistochemical detection of CXCL5 in human frontal white matter vasculature in subjects with cerebrovascular pathology (C). Percentage of CXCL5+ vessel segments in peri-ventricular white matter (n = 10) (p = 0.0005). Error bars represent S.E.M. Scale bar: 10 μm

Article Snippet: A plasmid containing the open reading frame of the murine CXCL5 sequence with a 3′ stop codon was purchased from Origene (#MR200761).

Techniques: Clinical Proteomics, Modification, Immunohistochemical staining

Journal: Cell reports

Article Title: IL-17/CXCL5 signaling within the oligovascular niche mediates human and mouse white matter injury

doi: 10.1016/j.celrep.2022.111848

Figure Lengend Snippet:

Article Snippet: A plasmid containing the open reading frame of the murine CXCL5 sequence with a 3′ stop codon was purchased from Origene (#MR200761).

Techniques: Blocking Assay, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Luminex, Hybridization, Plasmid Preparation, Biomarker Discovery, Control, Software

Journal: Annals of the Rheumatic Diseases

Article Title: A homeostatic function of CXCR2 signalling in articular cartilage

doi: 10.1136/annrheumdis-2014-205546

Figure Lengend Snippet: CXCL6 is present in healthy articular cartilage and its expression is associated with chondrocyte differentiation. (A) Immunofluorescence staining for CXCL6 (green) in normal and early osteoarthritis (moderate Mankin score) articular cartilage. Nuclei are stained using propidium iodide (red). Scale bar, 100 μm. (B) Densitometric quantification of CXCL6 staining (n=3). (C) Immunofluorescence staining for CXCL6 (red) in mouse articular cartilage of sham-operated control and destabilisation of the medial meniscus (DMM) operated mice, with 4′,6-diamidino-2-phenylindole staining the nuclei. Scale bar, 100 μm. (D) Densitometric quantification of CXCL6 staining (n=4). (E) Western blot analysis of CXCL6 release into supernatant from vehicle control or heparitinase treated, freeze-thawed wild-type mouse hip caps. (F) Real-time RT-PCR for CXCL6 mRNA in early and late passage human articular chondrocytes (n=3), *** p<0.001 by paired t test. (G) Alcian blue staining and spectrophotometric quantification of ATDC5 cell micromasses differentiated for 14 days using insulin (n=6). (H) Real-time RT-PCR quantification of CXCL6 mRNA expression in ATDC5 cells following 14 days of culture in either control or insulin supplemented differentiation medium (n=6) **p<0.01, **** p<0.0001.

Article Snippet: Primary antibodies used were rabbit anti-mouse pAKT (ser473) (Cell Signaling) 1:200 dilution, rabbit anti-mouse AKT (Cell Signaling) 1:500 dilution or rabbit anti-mouse CXCL6 (Biorbyt) 1:200 dilution in blocking solution at 4°C overnight.

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Quantitative RT-PCR

Journal: Annals of the Rheumatic Diseases

Article Title: A homeostatic function of CXCR2 signalling in articular cartilage

doi: 10.1136/annrheumdis-2014-205546

Figure Lengend Snippet: CXCR2 modulation of the articular chondrocyte phenotype is mediated by AKT. (A) Western blot of phospho-AKT (ser473) in wild-type mouse chondrocytes following 30 min incubation with recombinant mouse CXCL6. (B) Western blot comparison of phospho-AKT in freshly isolated chondrocytes from wild-type and CXCR2 −/− mice. (C) Immunofluorescence staining for pAKT in mouse articular cartilage of unchallenged wild-type and CXCR2 −/− mice, nuclei are stained with 4′,6-diamidino-2-phenylindole. Scale bar, 100 μm. (D, E) Real-time RT-PCR analysis of SOX9 and COL2A1 mRNA expression of wild type and CXCR2 −/− early passage mouse chondrocytes 24 h following transfection with either a control empty plasmid or constitutively active AKT (caAKT) expressing plasmid. (F) Real-time RT-PCR analysis of COL2A1 mRNA expression of wild-type and CXCR2 −/− mouse chondrocytes 24 h following transfection with either a control empty plasmid or a SOX9 expressing plasmid, *p<0.05, **p<0.01, *** p<0.001.

Article Snippet: Primary antibodies used were rabbit anti-mouse pAKT (ser473) (Cell Signaling) 1:200 dilution, rabbit anti-mouse AKT (Cell Signaling) 1:500 dilution or rabbit anti-mouse CXCL6 (Biorbyt) 1:200 dilution in blocking solution at 4°C overnight.

Techniques: Western Blot, Incubation, Recombinant, Comparison, Isolation, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Transfection, Control, Plasmid Preparation

Journal: Annals of the Rheumatic Diseases

Article Title: A homeostatic function of CXCR2 signalling in articular cartilage

doi: 10.1136/annrheumdis-2014-205546

Figure Lengend Snippet: Disruption of CXCR2 signalling results in increased chondrocyte apoptosis in an AKT-dependent manner. (A) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of wild-type and CXCR2 −/− articular cartilage 8 weeks following destabilisation of the medial meniscus surgery. Scale bar, 100 μm. (B) Quantification of TUNEL-positive chondrocytes in superficial and deep zones of articular cartilage of wild-type and CXCR2 −/− mice (n=5). (C) TUNEL staining of monolayer differentiated ATDC5 24 h following co-transfection with either scrambled control or CXCR2 siRNA along with either a control or caAKT expressing plasmid. Scale bar, 100 μm. (D) Quantification of TUNEL-positive ATDC5 cells following siRNA and plasmid transfection (n=3) **p<0.01, *** p<0.001. (E) In healthy articular cartilage, CXCL6 is expressed by chondrocytes and retained within the extracellular matrix (ECM) by HSPGs where it is available and required for signalling via CXCR1 and CXCR2 on nearby chondrocytes for the maintenance of their phenotypic stability. During osteoarthritis, mechanical and inflammatory injury leads to the breakdown of HSPGs within the ECM, leading to the release of CXCL6. This not only results in the release of CXCL6 from the articular cartilage, but disrupts the cell-autonomous ELR+ CXC chemokine signalling mechanism required for chondrocyte homeostasis.

Article Snippet: Primary antibodies used were rabbit anti-mouse pAKT (ser473) (Cell Signaling) 1:200 dilution, rabbit anti-mouse AKT (Cell Signaling) 1:500 dilution or rabbit anti-mouse CXCL6 (Biorbyt) 1:200 dilution in blocking solution at 4°C overnight.

Techniques: Disruption, TUNEL Assay, Staining, Cotransfection, Control, Expressing, Plasmid Preparation, Transfection