Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 1. Immunofluorescence of CXCL12 and CXCR4 in spiral ganglion neurons of neonatal mouse inner ear. CXCL12 and CXCR4 expression in spiral ganglion neurons were detected by immunostaining at (A) P0, (B) P7, (C) P14 and (D) P21. Nuclei were stained with DAPI and merged images of red, green and DAPI staining are shown. Images were captured under a fluorescence microscope (magnification, x40). CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4; DAPI, 4'‑6‑diamidino‑2‑phenylindole dihydrochloride; P, postnatal day.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Immunofluorescence, Expressing, Immunostaining, Staining, Fluorescence, Microscopy

Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 2. Quantitative analysis of relative fluorescence intensity. Relative fluorescence intensity of (A) CXCL12 and (B) CXCR4 in the different groups, fol lowing normalization with the P0 group. Fluorescence intensity was quantified using ImageJ software. *P<0.05 and **P<0.01 vs. P0; &P<0.05 vs. P7. Data are presented as the mean ± standard deviation. CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4; P, postnatal day.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Fluorescence, Software, Standard Deviation

Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 3. mRNA and protein expression levels of CXCL12 and CXCR4 in spiral ganglion neurons of neonatal mice. (A) Relative expression levels of CXCL12 and CXCR4 mRNA on P0, P7, P14 and P21 detected by reverse transcription‑quantitative polymerase chain reaction analysis. (B) Concentration of CXCL12 protein was quantified by enzyme‑linked immunosorbent assay in the different groups. (C) Protein expression of CXCR4 in spiral ganglion neurons of neonatal mice detected by western blot analysis. (D) Relative protein expression was quantified using Image J software. *P<0.05 and **P<0.01 vs. P0; &P<0.05 vs. P7. Data are presented as the mean ± standard deviation. CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4; P, postnatal day.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Expressing, Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Software, Standard Deviation

Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 4. CXCL12 enhances the expression of CXCR4 in spiral ganglion neurons. (A) Reverse transcription‑quantitative polymerase chain reac tion analysis of CXCR4 mRNA expression in spiral ganglion neurons. Spiral ganglion neurons were treated with 100 ng/ml CXCL12, with or without 20 µg/ml AMD3100 for 7 days. *P<0.05 vs. control group; &P<0.05 vs. CXCL12‑treated group. (B) Western blot analysis of CXCR4 protein expression in different treatment groups. (C) Relative protein expression level of CXCR4 was quantified using Image J software. *P<0.05 vs. con trol group; &&P<0.01 vs. CXCL12‑treated group. Data are presented as the mean ± standard deviation. CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Expressing, Control, Western Blot, Software, Standard Deviation

Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 5. CXCL12/CXCR4 signaling inhibits the apoptosis of spiral ganglion neurons. (A) Western blot analysis of caspase‑3 and cleaved caspase‑3 protein expression in spiral ganglion neurons. Spiral ganglion neurons were treated with 100 ng/ml CXCL12 with or without 20 µg/ml AMD3100 for 7 days. (B) Relative protein expression levels of caspase‑3 and cleaved caspase‑3 were quantified using Image J software. *P<0.05 vs. control, day 7; &P<0.05 vs. CXCL12‑treated group, day 7. Data are presented as the mean ± standard deviation. CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Western Blot, Expressing, Software, Control, Standard Deviation

Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 7. Photomicrographs illustrating spiral ganglion neurons (magnification, x40). Spiral ganglion neurons were stained by neuron‑specific enolase antibody in the (A) control and (B) CXCL12/CXCR4‑blocked groups. The mice were administered with CXCR4 antagonist, AMD3100, by intraperitoneal injection for 48 h. The spiral ganglion was then isolated and detected by immunohistochemistry using neuron‑specific enolase antibody. The spiral ganglion neurons exhibited a normal morphology in the control group, but were reduced in number and had a fuzzy cell morphology in AMD3100‑treated cells. Mice treated with phosphate‑buffered saline served as the control. CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Staining, Control, Injection, Isolation, Immunohistochemistry, Saline

Journal: Molecular medicine reports

Article Title: CXCL12/CXCR4 signaling pathway regulates cochlear development in neonatal mice.

doi: 10.3892/mmr.2016.5085

Figure Lengend Snippet: Figure 6. CXCL12/CXCR4 regulates the growth of dendrites in spiral ganglion neurons in vitro. (A) Western blot analysis of MAP2 protein expression in spiral ganglion neurons treated with CXCL12 in the presence or absence of 20 µg/ml AMD3100 for 7 days. (B) Relative protein expression level of MAP2 was quantified using Image J software. *P<0.05 vs. control, day 7; &P<0.05 vs. CXCL12‑treated group, day 7. (C) The mean number of dendrites per cell in the various treated groups. The mean number of dendrites were calculated on day 7. *P<0.05 vs. control; &P<0.05 vs. CXCL12‑treated group. (D) The mean length of dendrites per cell in the various treatment groups. *P<0.05 vs. control; &P<0.05 vs. CXCL12‑treated group. Data are presented as the mean ± standard deviation. MAP2, microtubule‑associated protein 2; CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4.

Article Snippet: The protein extract was centrifuged and the concentration of CXCL12 in the supernatant was determined using a commercially available CXCL12 ELISA kit (cat. no. CSB-E04723m; Cusabio Biotech Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: In Vitro, Western Blot, Expressing, Software, Control, Standard Deviation

Journal: Cell Death Discovery

Article Title: Fibroblast CEBPD/SDF4 axis in response to chemotherapy-induced angiogenesis through CXCR4

doi: 10.1038/s41420-021-00478-0

Figure Lengend Snippet: A SDF4 expression was induced following CEBPD induction by CDDP or 5-FU treatment in HFL1 cells infected with shβ-galactosidase (shC) or shCEBPD (shD) lentiviruses and treated with or without CDDP or 5-FU for 6 h. RT-PCR assays were conducted to examine the CEBPD , SDF4, SDF1 , and GAPDH transcript levels. B SDF4 expression was examined in conditioned medium (C.M.) or cell lysates from HFL1 cells after CDDP or 5-FU treatment for 6 h. p84 was used as an internal control. C CEBPD activates SDF4 reporter activity. Representation of reporter constructs (left panel). A reporter assay was conducted to assess the activity of the SDF4 reporter with or without CEBPD expression vector (right panel). D A ChIP assay was performed with the indicated antibodies. Three independent experiments were performed in triplicate. All data are expressed as the mean ± S.D. Differences between groups were analyzed with the unpaired two-tailed t -test. *** p < 0.001.

Article Snippet: The primary antibodies used in this study were mouse anti-CD31 monoclonal antibody (1:100 dilution; Zhongshan JinQiao, ZM-0044, China), rabbit anti-SDF4 polyclonal antibody (1:80 dilution; Proteintech, 10517-1-AP, USA), and mouse anti-VEGF-D monoclonal antibody (1:50 dilution; R&D systems, MAB286, USA).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay, Construct, Reporter Assay, Plasmid Preparation, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Fibroblast CEBPD/SDF4 axis in response to chemotherapy-induced angiogenesis through CXCR4

doi: 10.1038/s41420-021-00478-0

Figure Lengend Snippet: A The proliferation of HUVECs cultured in conditioned medium from HFL1 cells infected with shβ-galactosidase (shC) or shSDF4 (shS) lentiviruses and treated with or without CDDP or 5-FU for 6 h, followed by 18-h recovery with fresh medium was assessed. B The migration of HUVECs was assessed by determining the number of HUVECs cultured in conditioned medium from HFL1 cells infected with shC and shS lentiviruses and treated with or without CDDP or 5-FU for 6 h, followed by 18-h recovery with fresh medium. C Angiogenesis was assessed by counting the intersection number between branches of HUVECs growing in conditioned medium from HFL1 cells infected with shC and shS lentiviruses and treated with or without CDDP or 5-FU for 6 h, followed by 18 h recovery with fresh medium. D Assays to assess migration and in vitro tube formation were conducted as described in materials and methods. HUVECs were treated with SDF4 at 0.25, 0.5, and 1 μg/ml. E Cebpd +/+ (WT) mice were subcutaneously inoculated with 0.5-μg/ml SDF4, 1-μg/ml SDF4, or 0.2-μg/ml VEGF Matrigel plugs. The experimental mice were sacrificed, and the Matrigel plugs were removed to assess the newly formed blood vessels; hemoglobin levels were measured in the plugs using a Drakin’s reagent kit ( n = 3 per group). VEGF was used as a positive control. F A549 cells mixed with HFL1 cells carrying a shβ-galactosidase knockdown vector with GFP (shC-GFP) or a SDF4 knockdown vector with GFP (shS-GFP) were inoculated subcutaneously into the dorsal rear flanks of NOD-SCID mice, and the mice were treated with or without CDDP (5 mg/kg). The mice with A549-xenografted tumors were sacrificed in the 12th week. Tumor tissues were stained for CD31 (red) and GFP (green), and nuclei were stained with DAPI (blue). G The number of metastasis nodules from A549-xenografted tumors in the lungs was determined. H Tumor volume was measured with external calipers and calculated using the standard formula: V = ( w × l 2 ) × 0.52, where l is the length and w is the width of the tumor ( n = 6 per group). Three independent experiments were performed in triplicate. All data are expressed as the mean ± S.D. Differences among groups were analyzed with one-way ANOVA followed by the Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used in this study were mouse anti-CD31 monoclonal antibody (1:100 dilution; Zhongshan JinQiao, ZM-0044, China), rabbit anti-SDF4 polyclonal antibody (1:80 dilution; Proteintech, 10517-1-AP, USA), and mouse anti-VEGF-D monoclonal antibody (1:50 dilution; R&D systems, MAB286, USA).

Techniques: Cell Culture, Infection, Migration, In Vitro, Positive Control, Knockdown, Plasmid Preparation, Staining, Comparison

Journal: Cell Death Discovery

Article Title: Fibroblast CEBPD/SDF4 axis in response to chemotherapy-induced angiogenesis through CXCR4

doi: 10.1038/s41420-021-00478-0

Figure Lengend Snippet: A Cell membrane fractions were incubated with or without GST-SDF4, and then, a co-immunoprecipitation assay was performed with antibody against SDF4, GST or CXCR4. B An immunofluorescence assay was conducted to assess GST, SDF4, and CXCR4 signals. C Angiogenesis was assessed by counting the branched intersection number of SDF4-treated HUVECs with or without AMD3100 treatment as indicated. D , E The activity of AKT1, ERK1/2, and p38 in response to SDF4 (0.5 μg/ml) and with or without AMD3100 (20 μg/ml) treatment in the indicated time courses. F Angiogenesis was assessed by counting the branched intersection number of HUVECs treated with SDF4 and/or the indicated kinase inhibitors or SDF4 combined with pretreatment with increasing concentrations of wortmannin, PD98059 or SB203580. G VEGFD expression in response to various signaling inhibitors on SDF4-treated HUVECs. Three independent experiments were performed in triplicate. H LLC1-Luc2 cells were orthotopically inoculated into the lung of C57BL/6 mice. The experimental mice were treated with CDDP or AMD3100 as indication after inoculation with tumor cells. Representative in vivo bioluminescent images and total tumor flux of LLC1-Luc2-bearing mice in each group shown at 5th week. n = 8 per group. All data are expressed as the mean ± S.D. Differences among groups were analyzed with the one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used in this study were mouse anti-CD31 monoclonal antibody (1:100 dilution; Zhongshan JinQiao, ZM-0044, China), rabbit anti-SDF4 polyclonal antibody (1:80 dilution; Proteintech, 10517-1-AP, USA), and mouse anti-VEGF-D monoclonal antibody (1:50 dilution; R&D systems, MAB286, USA).

Techniques: Membrane, Incubation, Co-Immunoprecipitation Assay, Immunofluorescence, Activity Assay, Expressing, In Vivo, Comparison

Journal: Cell Death Discovery

Article Title: Fibroblast CEBPD/SDF4 axis in response to chemotherapy-induced angiogenesis through CXCR4

doi: 10.1038/s41420-021-00478-0

Figure Lengend Snippet: A Tissue specimens from 57 patients with lung cancer treated with or without cisplatin were stained for SDF4 expression. SDF4 was positively correlated with fibroblasts in cisplatin-treated lung cancer patients. B , C SDF4, VEGF-D, and CD31 staining was performed via IHC with their individual specific antibodies. SDF4 abundance was correlated with VEGF-D and CD31 signals in 57 lung cancer patients. D Correlation between SDF4 abundance and the survival rate of cisplatin-treated lung cancer patients. Differences between patient subsets in overall survival were determined via Kaplan–Meier plot analysis and log-rank tests. A two-tailed p < 0.05 was considered statistically significant. Statistical analysis was performed with SPSS v. 17.0 software (SPSS, USA).

Article Snippet: The primary antibodies used in this study were mouse anti-CD31 monoclonal antibody (1:100 dilution; Zhongshan JinQiao, ZM-0044, China), rabbit anti-SDF4 polyclonal antibody (1:80 dilution; Proteintech, 10517-1-AP, USA), and mouse anti-VEGF-D monoclonal antibody (1:50 dilution; R&D systems, MAB286, USA).

Techniques: Staining, Expressing, Two Tailed Test, Software