Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 1. CXCR4+ DTC cells exhibit enhanced self-renewal activity, tumorigenic potential and IR resistance compared to CXCR4- cells. (a) MTT assay, (b) sphere-forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR cl, 22onogenic survival assay (black bar 50 mm), and (f) Western blot (left) and RT-PCR (right) analyses of DTC cells after sorting with apc-conjugated CXCR4 antibody. *p < .05. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: Activity Assay, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 2. Silencing of CXCR4 via shRNA infection suppresses self-renewal activity, tumorigenic potential and IR resistance in DTC cells. (a) MTT assay, (b) sphere-forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR clonogenic survival assay (black bar 50 mm), (f) Western blot (left) and RT-PCR (right), and (g) immunofluorescence (white bar 100 μm) analyses of DTC cells infected with shControl (shCTL), shCXCR4 a or D (upper) and quantification of the results (lower). *p < .05, **p < .01, ***p < .001. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: shRNA, Infection, Activity Assay, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 5. CXCR4 overexpressing U-CH1 cells exhibited higher self-renewal activity, tumorigenic potential and IR resistance than control cells. (a) MTT assay, (b) sphere- forming assay (white bar 50 mm), (c) limiting dilution assay, (d) soft agar assay (white bar 50 μm), (e) IR clonogenic survival assay (black bar 50 mm), (f) Western blot (left) and RT-PCR (right), and (g) immunofluorescence (white bar 100 μm) analyses of DTC cells of transduced with control (pQCXIP) and CXCR4 carrying retroviruses in U-CH1 cells (upper) and quantification of the results (lower). *p < .05, **p < .01. All experimental results were obtained from at least three independent experiments.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: Activity Assay, Control, MTT Assay, Limiting Dilution Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Transduction

Journal: Cancer biology & therapy

Article Title: CXCR4 confers stemness and radioresistance in chordoma cells.

doi: 10.1080/15384047.2025.2471631

Figure Lengend Snippet: Figure 6. Suppression of CXCR4 reduced tumorigenic potential and enhanced IR sensitivity of DTC cell in an in vivo xenograft model. (a) A representative image of tumor-bearing mice (left) and measurement of in vivo tumor growth rate (right) in shControl (shCTL) and shCXCR4-infected DTC cells subcutaneously transplanted in mice. (b) Combination therapy of IR and CXCR4 inhibitor (AMD3100, 5 mg/kg) in xenograft mouse model. (c) Immunohistochemistry (white bar 50 μm) of xenograft tissues probed with Sox2 antibody (left) and quantitation of the results (right) *p < .05, ***p < .001.

Article Snippet: To sort CXCR4+ and CXCR4− cells, DTC cells were dissociated into single cells, washed with ice-cold PBS, and stained with an APC-conjugated CXCR4 antibody (#130-100-070; Miltenyi Biotec Inc., Auburn, CA, USA) in 0.1 ml of binding buffer (2% BSA and 5 mm EDTA in PBS) at a 4°C refrigerator for 1 h. After washing with ice-cold PBS three times, cells were incubated with anti-APC microbead in 0.1 ml of binding buffer at a 4°C refrigerator for 30 min.

Techniques: In Vivo, Infection, Immunohistochemistry, Quantitation Assay

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A&B) The effect of IL-1β on the mRNA expression of chemokine receptors in Tca8113 (A) and Hep2 (B) cells. Cells were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4, CCR6 and CCR7 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) Quantitative CXCR4 mRNA expression in (A). * P < 0.05 compared with the non-treated group. (D) Time course of CXCR4 mRNA expression in response to IL-1β stimulation. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of CXCR4 were detected by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) The quantitative data corresponding to (D). * P < 0.05 compared with the non-treated group. (F) The effect of IL-1β on CXCR4 protein expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. CXCR4 protein expression was detected by FACS. (G) The effect of IL-1β on SDF-1α-induced cell migration. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. Cell migration in response to medium or 20 ng/ml SDF-1α was measured by the Transwell assay. * P < 0.05 compared with control groups. (I) The transwell assay showed cell migration in response to 20 ng/ml SDF-1α after treatment with the indicated concentrations of IL-1β for 24 h (Scale bars: 200 μM).

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Migration, Transwell Assay, Control

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The expression of IL-1 receptors in Tca8113 cells. Cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1R1 and IL-1RII were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) The expression of IL-1 receptors in Hep2. Cells were treated as described in (A). The mRNA levels of IL-1R1 and IL-1R2 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) The effect of IL-1β on IL-1R1 expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The protein levels of IL-1R1 were measured by western blot. β-actin protein levels were measured as loading controls. (D&E) The effect of IL-1Ra on IL-1β-induced CXCR4 mRNA up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR (D) and qRT-PCR (E). * P < 0.05 compared with the IL-1β-treated group. (F) The effect of IL-1Ra on IL-1β-induced CXCR4 protein up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 24 h. The protein expression of CXCR4 was measured by FACS. (G) The effect of RNA interference on the expression of IL-1R1 protein. Tca8113 cells, transfected with non-specific shRNA (Nssi) or with IL-1R1 shRNA (IL-1R1si), were treated with the indicated concentrations of IL-1β for 24 h. The expression of IL-1R1 protein was measured by western blot. β-actin protein levels were measured as loading controls. (H) The effect of IL-1R1 down-regulation on CXCR4 mRNA expression. Non-specific shRNA (Nssi) or IL-1R1 shRNA (IL-1R1si) transfected Tca8113 cells were treated with medium or 20 ng/ml IL-1β for 24. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The quantitative data corresponding to (H). * P < 0.05 compared with the Nssi group.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Transfection, shRNA

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on mRNA levels of IL-1β and TNF-α. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1β and TNF-α were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) IL-1β quantitative mRNA levels in (A). * P < 0.05 compared with the non-treated control. (C) Time-course of IL-1β mRNA expression in response to IL-1β treatment. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) IL-1β quantitative mRNA levels in (C). * P < 0.05 compared with the non-treated group. (E) The effect of IL-1Ra on IL-1β-induced IL-1β mRNA expression. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were stimulated with 20 ng/ml IL-1β for 24 h. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) IL-1β quantitative mRNA levels in (E). * P < 0.05 compared with the non-treated group. (G) The sustained effect of IL-1β on the expression of IL-1β and CXCR4. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated number of days. The mRNA levels of IL-1β and CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (H-I) Quantitative data of IL-1β (H) and CXCR4 (I) in (G). * P < 0.05 compared with the non-treated groups.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) Time-dependent activation of Notch by IL-1β. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. Activated Notch NCID fragments were detected by western blot. β-actin protein levels were measured as loading controls. (B) Dose dependent activation of Notch by IL-1β treatment for 1 h. (C) The effect of IL-1β on Hes1 mRNA levels. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of the Notch1 targeting gene Hes1 were measured by qRT-PCR. * P < 0.05 compared with the control group. (D) The effect of Notch inhibition on CXCR4 expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) Quantitative data of CXCR4 expression in (D). * P < 0.05 compared with IL-1β-treated alone group. (F) The effect of Notch inhibition on IL-1β expression induced by IL-1β. Cells were treated as described in (D). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (G) Quantitative data of IL-1β expression in (F). * P < 0.05 compared with the IL-1β-treated alone group. (H) The effect of Notch inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of Notch inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of the Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Control, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on the activation of MAPKs. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The phosphorylation levels of MAPKs were measured by western blot. β-actin protein levels were measured as loading controls. (B) The effect of IL-1β on the activation of IκB-α. Tca8113 cells were treated as described in (A). IκB-α protein levels were measured by western blot. β-actin protein levels were measured as loading controls. (C) The effect of ERK inhibition on IL-1β-induced CXCR4 expression. Tca8113 cells, pre-treated with the indicated concentrations of U0126 for 30 min, were stimulated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) Quantitative data of (C). * P < 0.05 compared with the IL-1β-treated group. (E) The effect of ERK inhibition on IL-1β-induced IL-1β mRNA expression. Tca8113 cells were treated as described in (C). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) Quantitative data of (E). * P < 0.05 compared with the IL-1β-treated group. (G) The effect of ERK inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of ERK inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: European journal of immunology

Article Title: Up-regulation of leukocyte CXCR4 expression by sulfatide: an L-selectin-dependent pathway on CD4+ T cells.

doi: 10.1002/eji.200737118

Figure Lengend Snippet: Figure 1. Sulfatide induces surface CXCR4 up-regulation on human blood leukocytes. Cells were isolated from peripheral blood, incubated with sulfatide (100 lg/mL) for 1 h at 37C and stained for CXCR4. (A) Flow cytometry histogram of a representative experiment. The dotted line, solid line and grey area represent respectively the isotype control, CXCR4 baseline level, and CXCR4 level after sulfatide treatment. (B, C) Summarized results of multiple experiments. Data is presented as mean fluorescence intensity relative to the basal expression (white and black columns represent respectively the control and sulfatide treatment while bars indicate SEM, n = 4). * p value <0.05, ** p value <0.01.

Article Snippet: PE-conjugated isotype control and PE- and FITC-conjugated anti-CXCR4 (12G5) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Incubation, Staining, Flow Cytometry, Control, Fluorescence, Expressing

Journal: European journal of immunology

Article Title: Up-regulation of leukocyte CXCR4 expression by sulfatide: an L-selectin-dependent pathway on CD4+ T cells.

doi: 10.1002/eji.200737118

Figure Lengend Snippet: Figure 3. Sulfatide-induced CXCR4 up-regulation is critically mediated by L-selectin in CD4+ T cells. (A) L-Selectin was cross-linked with Mel-14 and secondary antibodies; then, CXCR4 surface levels were measured (white and black columns represent the control and cross-linking treatments, respectively, while bars indicate SEM; n = 3). L-Selectin cross-linking induced CXCR4 up-regulation of murine spleen subsets. K562 cells were incubated with sulfatide (B) or Lam1-116 cross-linking antibody (C) (white and black columns represent the control and treatment groups, respectively; the striped column represents transfected CXCR4 up- regulation as measured by anti-HA tag; the bars indicate SEM; n = 3). Surface CXCR4 expression on K562 cells was up-regulated by sulfatide or Lam1-116 only in the presence of WT L-selectin (WT) and not in the presence of a mutant (~16) that lacks 16 of 17 intracellular amino acids, or the parent cells which do not have L-selectin. (D) Galactosylcerebroside (GalCer) has the same structure as sulfatide, but differs only by a sulfate group, as marked by arrow. (E) Galactosylcerebroside had no effects on CXCR4 up-regulation of CD4+ T cells when compared to no treatment (NT) (n = 3). (F) Splenocyte subsets were isolated from WT and L-sel–/– animals, incubated with sulfatide, then stained for CXCR4. The dotted line, solid line and grey area represent respectively the isotype control, CXCR4 baseline level, and CXCR4 level after sulfatide treatment. The histogram is representative of multiple experiments. Sulfatide induced CXCR4 up-regulation on CD8+ T cells and B cells independently of L-selectin, while it was essential on CD4+ and CD4+CD25+ T cells (n = 3). Galactosylcerebroside had no effects on any cells.* p value <0.05.

Article Snippet: PE-conjugated isotype control and PE- and FITC-conjugated anti-CXCR4 (12G5) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Control, Incubation, Transfection, Expressing, Mutagenesis, Isolation, Staining

Journal: European journal of immunology

Article Title: Up-regulation of leukocyte CXCR4 expression by sulfatide: an L-selectin-dependent pathway on CD4+ T cells.

doi: 10.1002/eji.200737118

Figure Lengend Snippet: Figure 5. Sulfatide-induced CXCR4 up-regulation has signaling and functional consequences for CD4+ T cells. (A) CD4+ T cells were incubated with sulfatide for various times and stained for CXCR4. CXCR4 was up-regulated rapidly; 50% of the maximal up- regulation was reached as early as after 2 min while galactosylcerebroside had no effect at any time point. (B, C) CD4+ T cells were incubated with sulfatide (S) or galactosylcerebroside (G), then stimulated with CXCL12 (200 ng/mL) for 30 s. Western blot analysis showed increased CXCL12-induced tyrosine phosphorylation (B) and PI3K activation (C) after exposure to sulfatide. Blots were quantified and expressed as relative intensity (n = 3). (D) CD4+ T cells were incubated with sulfatide or galactosylcerebroside (GalGer) and loaded into the top chamber of a Transwell plate with either CXCL12 (black) or CCL5 (stripes) in the bottom chamber for 2 h. Migration to CXCL12 was enhanced by exposure to sulfatide but not galactosylcerebroside or no treatment (NT). The migration to CCL5 or to no chemokine (white) was not affected. * p value <0.05.

Article Snippet: PE-conjugated isotype control and PE- and FITC-conjugated anti-CXCR4 (12G5) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Functional Assay, Incubation, Staining, Western Blot, Phospho-proteomics, Activation Assay, Migration

Journal: Natural Product Communications

Article Title: The Effects of Resveratrol Targeting MicroRNA-4325P/PDGF-B to Regulate Tumor Angiogenesis in Osteosarcoma Microenvironment

doi: 10.1177/1934578x231156705

Figure Lengend Snippet: Figure 10. Angiogenesis is promoted in the sprouting phase of CAM by miR-432-5p/PDGFB signaling. After exposure to MG-63 cells or HOS cells containing coverslip, 6-day-old embryos’ CAM was excised and subjected to RT-PCR analysis. TEM8, CALD1, CXCR4, Tie-2 and Ephrin-B2 have been identified as angiogenesis-associated genes. The expression of β-actin mRNA was used to normalize the expression of these mRNAs. The results are expressed as the mean ± SD. *P < 0.05 & **P < 0.01 represent a significant decrease when compared to the control or inhibitor. One-way ANOVA followed by Tukey’s multiple comparison was used to analyze the data.

Article Snippet: Twenty microgram of isolated protein was then utilized or Western blot analyzed utilizing antibodies against PDGFB (1:1200, MAB2201, Biotechne, R&D systems, USA), Tie-2 (1:800; Biotechne R&D Systems, USA), Cycline A (1: 1000; Biotechne R&D Systems), Cdk1 (1:1000, Sigma Aldrich, USA), Cald1 (1:1000; Sigma Aldrich, USA), caspase-3 (Cell Signaling Technology, USA), Ephrin-B2 (1:800; Biotechne R&D systems, USA), CXCR4 (1:800; FAB170P, Biotechne R&D systems, USA), and α-tubulin (1:1200; Sigma Aldrich, USA) and CDK-2 (1:1200, Santa Cruz Biotechnology, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Comparison

Journal: Nature communications

Article Title: Neural progenitor cells orchestrate microglia migration and positioning into the developing cortex.

doi: 10.1038/ncomms6611

Figure Lengend Snippet: Figure 1 | Microglia distribution into the cerebral cortex overlaps Cxcl12 þ cortical domains. (a) Low magnification of the cerebral cortex at E10.5. High magnification (arrow) is shown in b (n ¼ 3). Labelling for Iba1 and CD45 in the cortical wall shows double-positive cells in the cortex and in the meninges (arrows indicate Iba1/CD45 þ cells). (c,d) Absolute numbers and cell densities of Iba1 þ cells were calculated by stereology at E14.5 and E18.5, and means±s.e.m. (pallium indicates the cerebral cortex and the hippocampus, while the sub pallium indicates basal ganglionic eminences and the septum, n ¼ 6 for each group). (e) Representative E16.5 cortical section probed for Cxcl12, boxed area indicates the VZ/SVZ of the lateral cerebral cortex. (f,g) High magnification sections labelled for Tbr2 and Iba1 (n ¼ 5). (h) Cxcl12 þ and Iba1 þ cell distribution at the corticostriatal boundary and into the Dentate Gyrus at E16.5 (n ¼ 5). (i) Representative RT–PCR for CxcR7, CxcR4 and the housekeeping gene H3 performed on P2 brain-derived primary microglia cell culture (primary MG) and on E15.5-sorted CD45low/CD11b þ cells (sorted MG; n ¼ 3 independent experiments). (j) Representative plot of CxcR4 þ cells, gated on CD45 þCD11b þLy6c cells from E15.5 brains (n ¼ 3 independent experiments). (k) Double labelling for Iba1 and CxcR4 in the cortical wall of E15.5 brain. Scale bar represents 100 mm. A two-tailed t-test was used to assess statistical significance: **Po0.01, ***Po0.001.

Article Snippet: The following antibodies were used: rabbit a-CxcR4 (Novus) 1:100; rabbit a-Iba1 (Wako) 1:400; rabbit a-Tbr2 (Abcam) 1:500; chicken a-GFP (Abcam) 1:500; rabbit a-pH3 (Cell Signaling) 1:200; rat a-CD206 (BD) 1:50; rabbit a-Ki67 (Novocastra) 1:100; rabbit a-CaB (Swant) 1:500; rat a-CD31 (BD) 1:100; rat a-BrdU (Abcam) 1:500; mouse a-IdU (BD) 1:75; rat a-CD45 (BD) 1:100; rabbit a-Casp3a (BD) 1:400; mouse a-F4/80 (Abcam) 1:200; rabbit a-Blbp (Millipore) 1:700; and click-it EdU alexafluor 647 imaging reagent (Invitrogen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Cell Culture, Two Tailed Test

Journal: Nature communications

Article Title: Neural progenitor cells orchestrate microglia migration and positioning into the developing cortex.

doi: 10.1038/ncomms6611

Figure Lengend Snippet: Figure 2 | The overexpression of Cxcl12 recruits microglia into the VZ/SVZ. Confocal images of E16.5 cortices receiving GFP plasmids (a,c,f,h) or GFP/Cxcl12 plasmids (b,d,e,g,i). (a,b) Double labelling for Blbp, GFP. (c,d) Double labelling for Tbr2 and GFP. (e) Ectopic Cxcl12 expression in the VZ/SVZ of Cxcl12-electroporated brains (n ¼ 5 for each group). (f,g) Distribution of Casp3a in GFP and GFP/Cxcl12-electroporated brains. (h,i) Confocal images of GFP (green), EdU (violet) and Iba1(red) in electroporated embryos pulsed with EdU for 3 h before killing. (j) Iba1 þ cells (means±s.e.m.) and Iba1/XdU þ cells (percentages±s.e.m.) in boxed areas (k; n ¼ 5 brains for each group). (l) Chemotactic responses of primary microglia to increasing amount of Cxcl12 and its reversion by AMD3100 (n ¼ 3). (m,n) Iba1 detection in E13.5 coronal sections from brains injected either with PBS (m) or AMD3100 (n). Cell counts (mean±s.e.m) are provided in o, (n ¼ 3 brains for each group). (p,q) Iba1 þ cells in E14.5 coronal sections from CxcR4 þ / and from CxcR4 / brains and their means±s.e.m. in r (n ¼ 3 brains for each group). Scale bar represents 100 mm. A two-tailed t-test was used to assess statistical significance: *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: The following antibodies were used: rabbit a-CxcR4 (Novus) 1:100; rabbit a-Iba1 (Wako) 1:400; rabbit a-Tbr2 (Abcam) 1:500; chicken a-GFP (Abcam) 1:500; rabbit a-pH3 (Cell Signaling) 1:200; rat a-CD206 (BD) 1:50; rabbit a-Ki67 (Novocastra) 1:100; rabbit a-CaB (Swant) 1:500; rat a-CD31 (BD) 1:100; rat a-BrdU (Abcam) 1:500; mouse a-IdU (BD) 1:75; rat a-CD45 (BD) 1:100; rabbit a-Casp3a (BD) 1:400; mouse a-F4/80 (Abcam) 1:200; rabbit a-Blbp (Millipore) 1:700; and click-it EdU alexafluor 647 imaging reagent (Invitrogen).

Techniques: Over Expression, Expressing, Injection, Two Tailed Test