Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.

doi: 10.4049/jimmunol.177.12.8730

Figure Lengend Snippet: FIGURE 1. MIF signaling components in prostate epithelial cells. A, Representative Western blot bands. Cell lysates (10 g of total protein) from 80% confluent cells grown under standard conditions were separated by SDS-PAGE under reducing conditions and transferred to PVDF membranes. Western blots were developed using anti-human MIF, anti-human CD74, or anti-human CD44v9 Abs. -Actin was used as a loading control. Figures depict representative samples from duplicate experiments displayed as composites of multiple Western blots. B, Analysis of MIF signaling components in epithelial cells. Prostate cancer cell lines (LNCaP and DU-145) demonstrate increased protein content of all components of MIF signaling (p 0.001) compared with benign prostate epithelial cells (PrEC and BPH-1). All band intensities were normalized to -actin. Significant difference compared with PrEC is represented by asterisks as follows: , p 0.01; , p 0.001. Values represent the mean SEM of duplicate measurements. Results are representative of duplicate experiments. C, Coimmunoprecipitation of CD74 and CD44v9 with MIF in prostate cancer cells. Cell lysates from 80% confluent cells (200 g of total protein) were immunoprecipitated with MIF polyclonal Ab and the protein G-binding protein complexes were separated by SDS-PAGE followed by Western blotting using anti-CD74 or CD44v9 Abs. Figures depict representative samples from duplicate experiments displayed as composites of multiple Western blots. D, Cell surface expression of MIF and CD74 in prostate epithelial cells. Biotinylation: DU-145 cells express increased total cell surface proteins. BPH-1 and DU-145 cell surface proteins were labeled with biotin. Avidin-purified cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and total biotinylated protein was detected using HRP-labeled streptavidin. Immunoprecipitation: DU-145 express increased cell surface MIF and CD74 compared with BPH-1 cells. Avidin-purified biotinylated cell lysates were immunoprecipitated with mono- clonal MIF, CD44v9, or monoclonal CD74 Ab. The resulting immunoprecipitate was separated by SDS-PAGE, transferred to PVDF membranes, and total biotinylated protein was detected using HRP-labeled streptavidin. Figures depict representative samples from duplicate experiments displayed as composites of multiple Western blots.

Article Snippet: Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose.

Techniques: Western Blot, SDS Page, Control, Immunoprecipitation, Binding Assay, Expressing, Labeling, Avidin-Biotin Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.

doi: 10.4049/jimmunol.177.12.8730

Figure Lengend Snippet: FIGURE 3. Effect of blocking MIF or its receptor (CD74) on prostate epithelial cell proliferation. A, Viability of BPH-1, LNCaP, and DU-145 cells treated with isotype control or anti-MIF Ab were determined by WST-1 assay. At each time point, 10 l of WST-1 reagent (Pierce) was added to the culture medium and incubated at 37°C for 3 h. Percentage of control cells was calculated by dividing each treatment well A450 by mean isotype control well A450. Data were determined from triplicate wells. Each point represents triplicate wells of mean percentage of WST-1 A450 SEM where significant difference compared with isotype control is represented by asterisks as follows: , p 0.05; , p 0.001. Results are mean SEM of three separate experiments. B, Viability of BPH-1, LNCaP, and DU-145 cells treated with anti-CD74 Ab was determined by WST-1 assay as described above. Significant difference compared with isotype control is represented by asterisks as follows: , p 0.01; , p 0.001. Results are mean SEM of three separate experiments.

Article Snippet: Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose.

Techniques: Blocking Assay, Control, WST-1 Assay, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.

doi: 10.4049/jimmunol.177.12.8730

Figure Lengend Snippet: FIGURE 4. Blocking MIF or CD74 induces DU-145 cell apoptosis. A, DNA laddering was observed in DU-145 cells treated with anti-MIF or anti-CD74 (5 g/ml) for 48 h. Two micrograms of genomic DNA from isotype control, anti-MIF or anti-CD74 Ab treated BPH-1, LNCaP, or DU- 145 cells was run on 1.5% agarose gel stained with ethidium bromide and photographed under UV light. DNA markers were electrophoresed with a kilobase pair reference with band range from 10 to 0.5 kb (lane M). Figures depict representative samples from duplicate experiments displayed as composites of multiple gels. B, Caspase-3 activity was observed in DU-145 cells treated with MIF or CD74 RNAi. BPH-1, LNCaP, or DU-145 cells were preincubated with either scramble control, MIF, or CD74 RNAi for 48 h, washed and lysed, and total cellular protein was assayed for caspase- 3-like activity by measuring the release of pNA from the colorimetric caspase-3 substrate z-DEVD-pNA. Treatment with MIF or CD74 RNAi increased constitutive caspase-3 activity in DU-145 cells. Each bar repre- sents mean A405 nm SEM from triplicate wells. Significant difference compared with scramble control is indicated by asterisks as follows: , p 0.01. Results are mean SEM of two separate experiments.

Article Snippet: Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose.

Techniques: Blocking Assay, DNA Laddering, Control, Agarose Gel Electrophoresis, Staining, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.

doi: 10.4049/jimmunol.177.12.8730

Figure Lengend Snippet: FIGURE 6. Effect of RNAi on MIF and CD74 expression and cell pro- liferation in DU-145 cells. DU-145 cells were treated with RNAi with samples taken at 24-h intervals. Gene expression was assessed by real-time RT-PCR. Total cell lysates were used to determine MIF and CD74 protein amounts by Western blots. Cell proliferation was assessed by WST-1 assay with each point representing mean percentage of WST-1 A450 SEM of triplicate wells. Significant difference compared with RNAi scramble con- trol is represented by asterisks as follows: , p 0.01; , p 0.001. All results are representative of three separate experiments. A, MIF mRNA amounts in RNAi-treated DU-145 cells. B, CD74 mRNA amounts in RNAi-treated DU-145 cells. C, Total MIF protein detected by Western blot in MIF or CD74 RNAi-treated DU-145 cell lysates. D, Total CD74 protein detected by Western blot in MIF or CD74 RNAi-treated DU-145 cell ly- sates. E, Cell proliferation in RNAi-treated DU-145 cells.

Article Snippet: Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot, WST-1 Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.

doi: 10.4049/jimmunol.177.12.8730

Figure Lengend Snippet: FIGURE 5. Blocking MIF or CD74 reduces in vitro invasion of DU-145 cells. Comparison of invading BPH-1, LNCaP, or DU-145 cells with MIF or CD74 Ab treatment. Invasion assays were performed in 24-well Trans- well chambers containing Matrigel-coated polycarbonate filters with 12 m pores. Invading cells in nine representative fields were counted under a microscope at 400 magnification. Each bar represents the mean number of invading cells SEM from triplicate filters of two separate experiments. Significant difference compared with isotype control is indicated by aster- isks as follows: , p 0.01.

Article Snippet: Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose.

Techniques: Blocking Assay, In Vitro, Comparison, Microscopy, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells.

doi: 10.4049/jimmunol.177.12.8730

Figure Lengend Snippet: FIGURE 7. p-ERK 1/2 amounts in BPH-1, LNCaP, or DU-145 cells treated with MIF or CD74 RNAi. A, ERK 1/2 Western blots. ERK 1/2 activation was examined by blotting with p-ERK 1/2 Ab (AF-1018; R&D Systems). The stripped membrane was reprobed with total ERK 1/2 Ab (AF-1576; R&D Systems). Figures depict representative samples from trip- licate experiments displayed as composites of multiple gels. B, p-ERK 1/2 was quantified by determining total p-ERK 1/2 band intensity and dividing by total ERK 1/2 band intensity for each sample. Data are expressed as percentage of control p-ERK 1/2 for each cell line. Values represent the mean value SEM of duplicate measurements of triplicate experiments. Significant difference compared with RNAi scramble control is represented by asterisks as follows: , p 0.001.

Article Snippet: Biotinylated proteins were detected using HRP-conjugated streptavidin and chemiluminescent substrate. mAbs were used in the immunoprecipitation of biotinylated proteins as follows: LN2 specific for the carboxy terminus of human CD74 was obtained from Santa Cruz Biotechnology (sc-6262), mAb specific for human MIF was obtained from R&D Systems (MAB289), and CD44v9 mAb (HB-258; ATCC) was purified from cell culture supernatant by protein G-agarose.

Techniques: Western Blot, Activation Assay, Membrane, Control

Journal: Journal of Translational Medicine

Article Title: Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

doi: 10.1186/1479-5876-6-21

Figure Lengend Snippet: UVB-induced apoptosis of TC-1 cells and maturation of TC-1 antigen-loaded DCs. ( A ) TUNEL assay of UVB-treated and control TC-1 cells 24 hours post-irradiation. ( B ) DNA ladder assay of TC-1 cells 24 hours after irradiation with UVB light. ( C ) Flow cytometry analysis of annexin V in UVB-treated cells 24 hours post-irradiation. ( D ) Percentage of DCs that have engulfed tumor cells, as determined by flow cytometry. DCs were pulsed with cells killed by UVB. Apoptotic tumor cells were labeled with PKH26 prior to pulsing. DCs were stained with antibody against CD11c.

Article Snippet: Apoptosis at 24 hours was quantified by flow cytometry detection of annexin-V staining using the TACSTM Annexin-Biotin Apoptosis detection kit (R&D Systems, Minneapolis, MN) and confirmed with the ApopTag peroxidase in situ detection kit (Intergen, Purchase, NY) and the Apoptotic DNA-Ladder Kit (Roche), according to the manufacturers' instructions.

Techniques: TUNEL Assay, Control, Irradiation, DNA Laddering, Flow Cytometry, Labeling, Staining