Journal: Molecular carcinogenesis

Article Title: SPI1-mediated CXCL12 expression in bladder cancer affects the recruitment of tumor-associated macrophages.

doi: 10.1002/mc.23663

Figure Lengend Snippet: FIGURE 1 Prognostic properties of C‐X‐C motif chemokine 12 (CXCL12) in bladder cancer (BC). (A) The survival of patients with BC differentially expressing CXCL12 using the Kaplan–Meier plotter database and GEPIA database. (B) Correlation analysis of CXCL12 with immune infiltration in BC analyzed using TIMER database. (C) TIMER2.0 database analysis of the correlation between CXCL12 in BC and M2 macrophage infiltration of tumor‐associated macrophages. (D) Detection of CXCL12 expression in BC cells and human uroepithelial cell line SV‐HUC‐1 by RT‐qPCR. All potency values are expressed as mean ± standard deviation of independent experiments in triplicate. Definition of statistical significance: *p < 0.05 according to one‐way analysis of variance.

Article Snippet: TAM chemotaxis assays were performed by adding 10 ng/mL or 50 ng/mL CXCL12 (460‐SD, R&D Systems) and incubating for 18 h at 37°C.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Molecular carcinogenesis

Article Title: SPI1-mediated CXCL12 expression in bladder cancer affects the recruitment of tumor-associated macrophages.

doi: 10.1002/mc.23663

Figure Lengend Snippet: FIGURE 2 Identification of in vitro tumor‐associated macrophages (TAMs). (A) Microscopic observation of the morphology of THP‐1 cells. (B) Observation of morphological changes of THP‐1 cells after phorbol 12‐myristate 13‐acetate (PMA) treatment. (C) Observation of the effects of interleukin (IL)‐4 and IL‐13 on cell morphology. (D–F) Detection of surface markers CD204 and CD206 in untreated (D), PMA−(E), and PMA + IL‐4/IL‐13‐treated (F) cells by flow cytometry. (G) Detection of C‐X‐C motif chemokine 12 (CXCL12) expression in J82 and T24 cells after si CXCL12 transfection by RT‐qPCR. (H) Chemotactic migration of TAMs after silencing and overexpression of CXCL12 were examined using migration assay. All potency values are expressed as mean ± standard deviation of independent experiments in triplicate. Definition of statistical significance: *p < 0.05 according to one‐way or two‐way analysis of variance.

Article Snippet: TAM chemotaxis assays were performed by adding 10 ng/mL or 50 ng/mL CXCL12 (460‐SD, R&D Systems) and incubating for 18 h at 37°C.

Techniques: In Vitro, Flow Cytometry, Expressing, Transfection, Quantitative RT-PCR, Migration, Over Expression, Standard Deviation

Journal: Molecular carcinogenesis

Article Title: SPI1-mediated CXCL12 expression in bladder cancer affects the recruitment of tumor-associated macrophages.

doi: 10.1002/mc.23663

Figure Lengend Snippet: FIGURE 5 SPI1 is an upstream factor of C‐X‐C motif chemokine 12 (CXCL12) in bladder cancer (BC) cells. (A) The intersection of transcription factors regulating CXCL12 predicting using the hTF‐target database and genes positively associated with CXCL12 in the BC downloaded from the UALCAN database. (B) Correlation analysis of SPI1 and CXCL12 in BC. (C) Kaplan–Meier analysis of survival in patients with BC differentially expressing SPI1. (D) Correlation analysis of SPI1 with immune infiltration in BC in the TIMER database. (E) Correlation between SPI1 expression and macrophage infiltration in BC analyzed by TISIDB database. (F) The binding site of the CXCL12 promoter to SPI1 was obtained from the Jaspar database. (G) Detection of SPI1 expression in J82 and T24 cells transfected with overexpression of SPI1 plasmid analyzed using RT‐qPCR. (H) SPI1 enrichment on CXCL12 promoter by chromatin immunoprecipitation. All potency values are expressed as mean ± standard deviation of independent experiments in triplicate. Definition of statistical significance: *p < 0.05 according to two‐way analysis of variance.

Article Snippet: TAM chemotaxis assays were performed by adding 10 ng/mL or 50 ng/mL CXCL12 (460‐SD, R&D Systems) and incubating for 18 h at 37°C.

Techniques: Expressing, Binding Assay, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Standard Deviation

Journal: Molecular carcinogenesis

Article Title: SPI1-mediated CXCL12 expression in bladder cancer affects the recruitment of tumor-associated macrophages.

doi: 10.1002/mc.23663

Figure Lengend Snippet: FIGURE 6 SPI1 mitigates the repressive effects of si C‐X‐C motif chemokine 12 (CXCL12) on bladder cancer (BC) cell biological activities and tumor‐associated macrophage recruitment. (A) Detection of SPI1 expression in BC cells and human uroepithelial cell line SV‐HUC‐1 by RT‐qPCR. (B) Detection of CXCL12 and SPI1 expression after si SPI1 transfection or co‐transfection (SPI1‐OE + si CXCL12) of cells by RT‐qPCR. (C) The proliferative activity of cells by CCK8. (D and E) The migration and invasion of cells by Transwell assays. (F) The apoptosis of cells by flow cytometry. All potency values are expressed as mean ± standard deviation of independent experiments in triplicate. Definition of statistical significance: *p < 0.05 according to one‐way or two‐way analysis of variance.

Article Snippet: TAM chemotaxis assays were performed by adding 10 ng/mL or 50 ng/mL CXCL12 (460‐SD, R&D Systems) and incubating for 18 h at 37°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Cotransfection, Activity Assay, Migration, Flow Cytometry, Standard Deviation

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: Pretreatment with CXCL12 promotes transition from G1 to S phase during growth factor deprivation (GFD). ( A-C ) Flow cytometry analysis shows the cell cycle phase of P13.5 mouse neural progenitor cells which were pretreated with CXCL12 for 2 hours and then treated without growth factors for 0 to 12 hours. ( D ) Results are presented as the mean ± SEM of three independent experiments with NPC from three mouse donors. *** P <0.001, significantly different from control group, ## P <0.01, ### P <0.001, significantly different from GFD group.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Flow Cytometry, Control

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: Real time PCR and Western blot analysis of CyclinD and β-catenin following CXCL12 pretreatment. ( A ) mNPCs pretreated with CXCL12 at different concentrations (0,10,50,100 ng/ml) for 2 hours and then treated without growth factors for 24 hours. Values are means ± SEM (n=3). ( B ) mNPC pretreated with CXCL12 at 0, 50, 100, 250 ng/ml for 2 hours following 24 hour starvation. Values are means ± SEM (n=3), * P <0.05, ** P <0.01, significantly different from the GFD group. ( C ) mNPCs were pretreated with CXCL12 for 2 hours and then treated without growth factors for 24 hours. ( D ) Levels of β-catenin were normalized as a ratio of β-catenin to actin after densimetrical quantification of panel C and shown as fold change relative to GFD group. Results are expressed as the mean ± SEM of triplicate samples and are representative of 3 independent experiments. * P <0.05, compared with GFD.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: Proliferation effect of CXCL12 on mouse NPC by Ki67 staining. CXCL12-mediated proliferation of mouse NPC is determined by Ki67 staining after pretreatment of increasing concentrations of CXCL12 stimulation for 2 hours followed by the deprivation of growth factors for 24 hours. Cell proliferation was assessed by immuno-staining for Ki67 positive in mNPC in GFD group ( A,B,C ) and with CXCL12 pretreatment group ( D,E,F ). The percentage of Ki67-positive proliferating mNPCs were determined for each treatment with different concentrations of CXCL12 by counting positive cells per microscopic field in 30 pictures per condition ( G,H ). Results are presented as the mean ± SEM of three independent experiments with NPC from three mouse donors. * P <0.05, ** P <0.01, in comparison to GFD group, scale bar=100 μm.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Staining, Immunostaining, Comparison

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: CXCL12 pretreatment protected mNPC against apoptosis induced by the deprivation of growth factors. mNPCs were pretreated with CXCL12 for 2 hours and then deprived of growth factors for 24 hours. mNPCs apoptosis was determined by TUNEL assay ( A-D ) or PARP cleavage assay ( F ). ( A-D ) Representative pictures from TUNEL assay are shown. ( E ) Quantification data of TUNEL assay were determined as a percentage of TUNEL positive cells against the total cell number. ( F ) Cleaved PARP protein levels were determined by Western blotting. ( G ) Levels of cleaved PARP were normalized as a ratio of cleaved PARP to actin after densimetrical quantification of panel F and shown as fold change relative to control. Results are expressed as the mean ± SEM of triplicate samples and are representative of 3 independent experiments. ## P <0.01 compared with control; * P <0.05, ** P <0.01 compared with growth factors deprivation group without CXCL12 pretreatment. Scale bar=100 μm.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: TUNEL Assay, Cleavage Assay, Western Blot, Control

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: CXCR7 is required for the proliferation function of CXCL12 in mNPCs. ( A-D ) immuno-staining for DAPI in CXCR4 knock out and CXCR7 knock out mNPCs in the starvation group ( A,C ) and with the CXCL12 pretreatment group ( B,D ). The total cell numbers were determined for each treatment with CXCL12 by counting positive cells per microscopic field in 30 pictures per condition ( E ). Results are presented as the mean ± SEM of three independent experiments with NPC from three mouse donors. ns>0.05 in comparison to starvation. Scale bar=100 μm.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Immunostaining, Knock-Out, Comparison