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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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: Cells of each group, which were stimulated by hypoxia, CXCL6 or BAY87-2243 (MCE, NJ) were used to extract total RNA with Trizol Reagent (TransStart, Beijing, China) following the manufacturer’s instructions.

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 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: 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