Journal: The Journal of Biological Chemistry

Article Title: Ets homologous factor (EHF) has critical roles in epithelial dysfunction in airway disease

doi: 10.1074/jbc.M117.775304

Figure Lengend Snippet: EHF depletion in Calu-3 cells alters secretion of a neutrophil chemokine. A, EHF ChIP qPCR confirmed its binding to sites near IL8, CXCL6, and CXCL1 in HBE cells. Shown is the average of all experiments with standard deviation (n = 4) (top panel). Also shown is a graphic of the Chr4q13.3 region with EHF ChIP-seq peaks (bottom panel). B and C, EHF was depleted (siRNA) in Calu-3 cells, followed by their exposure to carrier, IL-17a (B), or LPS (C). Gene expression was measured by RT-qPCR. β-actin (ACTB) was included as a negative control. **, p < 0.01 by two-way analysis of variance plus multiple comparisons test; ns, not significant (average with standard deviation). D and E, secretion of CXCL6 (D) and IL-8 (E) into medium conditioned by NC- and EHF-depleted Calu-3 quantified by colorimetric sandwich ELISA (n = 3). *, p < 0.05; paired two-tailed Student's t test. Bars show the average of all experiments, with error bars representing standard deviation.

Article Snippet: Quantikine colorimetric sandwich ELISA for IL-8 (D800C) and CXCL6 (DGC00) (both from R&D Systems) was performed according to the protocol of the manufacturer.

Techniques: Binding Assay, Standard Deviation, ChIP-sequencing, Expressing, Quantitative RT-PCR, Negative Control, Sandwich ELISA, Two Tailed Test

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 3. Effects of hypoxia on CXCL6 and CXCL2 gene expression in co-culture systems. (A) The difference of CXCL6 gene expression between censored (n ¼ 95) and relapse/refractory (n ¼ 247) patients in TARGET database. (B to E) The gene expression of CXCL6 and CXCL2 of MSCs and HUAECs in the co-culture systems were measured by the co-culture/mono culture ratio. (B and D) MSC þ THP-1/MSC; (C and E) HUAEC þ THP-1/HUAEC. *P < 0.05, **P < 0.01, ***P < 0.001, and “ns” for no statistical significance. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Gene Expression, Co-Culture Assay

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 4. CXCL6 and CXCL2 content in the supernatant of hypoxia and THP-1 co-culture systems. (A and B) CXCL6 content in supernatant. (C and D) CXCL2 content in supernatant. *P < 0.05, **P < 0.01, and ***P < 0.001. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Co-Culture Assay

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 5. The effects of CXCL6 and CXCL2 on the proliferation and migration of MSCs and HUAECs. (A and B) The effects of CXCL6 (A) and CXCL2 (B). Among them, (a and b) described the effects of cytokines on MSCs and (c and d) described the effects of cytokines on HUAECs. (a and c) showed the effects of different concentrations of cytokines on target cells. (b and d) showed the time-dependent effect of cytokines on target cells. (C) The chemotaxis of CXCL6 and CXCL2 to MSCs and HUAECs respectively. The concentration of CXCL6 was 50 ng/mL and of CXCL2 was 100 ng/mL. Scale ¼ 100 mm. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Migration, Chemotaxis Assay, Concentration Assay

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 6. Expression of cleaved-caspase3 in MSCs and HUAECs. (A) Expression of cleaved-caspase-3 (green fluorescence) measured by immunofluorescence method increased significantly in hypoxia (1% O2) for 48 h, but decreased after CXCL6 was supplied. The reversion depended on the concentration of CXCL6. MSCs were pictured at 20 water objective and HUAECs were pictured at 20 air objective. The heatmap represented the change of average fluorescence intensity in each group. (B) Western blot method verified the above results. The histograms (MSCs on the left and HUAECs on the right) represented the relative gray value of each group. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

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

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 7. Angiogenesis ability of vascular endothelial cells authenticated in vitro. (A) HUAECs in standard culture conditions, CoCl2 simulate hypoxia and hypoxia plus CXCL6 or CM conditions in vitro were used to simulated angiogenesis. HUVECs tubule forming experiment was taken as positive control. (B to E) Quantitative comparison of the number of master junction and meshes as well as total meshes area and total segments length. Scale ¼ 200 mm. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: In Vitro, Positive Control, Comparison

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 8. Correlation between CXCL6 gene expression and HIF-1a gene expression in AML patients. The original data were downloaded from Cbioporta (https:// www.cbioportal.org/datasets), n ¼ 323. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Gene Expression

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 9. Regulation of CXCL6 expression by HIF-1a. (A) MSCs or HUAECs were treated with CoCl2 (200 mmol/L), with or without BAY87-2243 (1 mmol/L), and the protein expression of HIF-1a was detected by Western blot method after 24 or 48 h. (B) The relative expression of HIF-1a in three independent experiments was statistically analyzed. The x-axis coordinates represented CoCl2()/BAY(), CoCl2(þ)/BAY(), CoCl2(þ)/BAY(þ), respectively. (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. (C) MSCs and HUAECs were cultured for 24 or 48 h under the condition of 1% O2, with or without BAY87-2243 (1 mmol/L). The relative expression of CXCL6 mRNA was detected by real-time PCR. (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 11. CXCL6 activate mTOR in MSCs and HUAECs. (A) CoCl2 (200 mmol/L) was used to treat MSCs or HUAECs for 24 h with or without CXCL6 of 50 ng/Ml. (B) The statistical results of three groups of independent experiments, **P < 0.01. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques:

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

doi: 10.1177/1535370220960675

Figure Lengend Snippet: Figure 10. Negative feedback regulation of CXCL6 on HIF-1a. (A) MSCs and HUAECs were cultured for 24 or 48 h under 1% O2 condition with or without CXCL6 (50 ng/mL). The relative mRNA expression of HIF-1a was detected by real-time PCR. (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. (B) MSCs or HUAECs were treated with CoCl2, with or without CXCL6 (50 ng/mL). After 24 or 48 h, the expression of HIF-1a protein was detected by Western blot method. (C) The relative expression of HIF-1a protein in three independent experiments was statistically analyzed, and the x-axis coordinates were respectively representative CoCl2()/CXCL6(), CoCl2(þ)/CXCL6(), CoCl2(þ)/CXCL6(þ), (a) MSC–24 h; (b) MSC–48 h; (c) HUAEC–24 h; (d) HUAEC–48 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and “ns” for no statistical significance. (A color version of this figure is available in the online journal.)

Article Snippet: The cytokine levels in the supernatant of each group were respectively detected by the human CXCL6 ELISA kit (BOSTER, Wuhan, China) and human CXCL2 ELISA Kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s protocol.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot

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 Neurology

Article Title: Early Microglial Activation Following Closed-Head Concussive Injury Is Dominated by Pro-Inflammatory M-1 Type

doi: 10.3389/fneur.2018.00964

Figure Lengend Snippet: Gene table for inflammation profile array genes and their TaqMan IDs.

Article Snippet: Rn00571440_m1 , Rn01430873_g1 , Rn00441826_m1 , Rn04181452_s1 , Rn02133647_s1 , Rn01488472_g1 , Rn01507024_m1 , Rn01471506_m1 , Rn00573587_g1 , Rn01767369_m1 , Rn01456850_m1.

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: 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: 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: 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 Assay, 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