Journal: The Journal of investigative dermatology

Article Title: Reduction in human epidermal Langerhans cells with age is associated with decline in CXCL14-mediated recruitment of CD14 + monocytes

doi: 10.1016/j.jid.2019.11.017

Figure Lengend Snippet: (a) Representative IF staining of CXCL14 (green) in young and old human skin tissues. Nuclei are stained with DAPI (blue) and dotted lines highlight the basement membrane (scale bar: 100 μm). (b) The quantitation of CXCL14+ keratinocytes per hpf image. (c) The quantitation of dermal CXCL14+ cells per hpf. (d) Correlation between the number of CXCL14+ keratinocytes and the number epidermal LCs across young and old skin samples. n = 20 in young group and n = 21 in old group, cells are counted blindly and averaged across 10 randomly selected hpf images per skin sample, all data are expressed as the mean ± SD, *** p < 0.0001, ns: not significant.

Article Snippet: In CXCL14 induction test, biopsied skin samples were injected subepidermally with 60 μL recombinant human CXCL14 (5 μg/mL, R&D systems, 866-CX) or PBS (carrier only) immediately before placing them in the top well of the transwell assay plates containing CFSE + THP-1 cells in the bottom chamber.

Techniques: Staining, Membrane, Quantitation Assay

Journal: The Journal of investigative dermatology

Article Title: Reduction in human epidermal Langerhans cells with age is associated with decline in CXCL14-mediated recruitment of CD14 + monocytes

doi: 10.1016/j.jid.2019.11.017

Figure Lengend Snippet: (a) The experimental scheme for ex vivo THP-1 migration into human skin. Nuclei are stained with DAPI (blue). The arrows point to CFSE+ THP-1 cells that have migrated into the dermis. Dotted line marks the basement membrane, scale bars: 100 μm. (b) The quantitation of CFSE+ THP-1 cells that have migrated into the skin per hpf image (n = 4 young and 5 old skin samples). (c) The effect of CXCL14 neutralizing antibody (50 μg/mL) treatment on the recruitment of CFSE+ THP-1 cells into young skin samples (n = 4 per group). Note that CXCL14 neutralizing antibody was added to the THP-1 media in the lower chamber of the transwell assay system. (d) The effect of CXCL14 recombinant protein (5 μg/mL) treatment on the recruitment of CFSE+ THP-1 cells into old skin samples (n = 4 per group). Note that recombinant human CXCL14 or PBS (carrier control) was injected subepidermally into the skin in the top well of the transwell assay system. Cells are counted blindly and averaged across 10 randomly selected hpf images per skin sample, all data are expressed as the mean ± SD, * p < 0.05.

Article Snippet: In CXCL14 induction test, biopsied skin samples were injected subepidermally with 60 μL recombinant human CXCL14 (5 μg/mL, R&D systems, 866-CX) or PBS (carrier only) immediately before placing them in the top well of the transwell assay plates containing CFSE + THP-1 cells in the bottom chamber.

Techniques: Ex Vivo, Migration, Staining, Membrane, Quantitation Assay, Transwell Assay, Recombinant, Control, Injection

Journal: Experimental neurology

Article Title: Cisplatin induces BDNF downregulation in middle-aged female rat model while BDNF enhancement attenuates cisplatin neurotoxicity

doi: 10.1016/j.expneurol.2024.114717

Figure Lengend Snippet: In vitro, ampakine CX546 prevents cisplatin-induced loss of BDNF expression, dendritic arborization, and PSD-95. (A) Cisplatin significantly reduced BDNF mRNA after exposure to 1 μM cisplatin at 24 h, which was prevented by co-treatment with 50 μM CX546. (B) Representative images of reconstructed neurons superimposed over 20 μm concentric Sholl circles, and (C) Sholl analysis quantification of mature hippocampal neurons exposed to 1 μM cisplatin with or without co-treatment of 50 μM CX546 for 24 h. (D) Representative images of dendritic branches immunostained for PSD-95 and (E) quantification of PSD-95 puncta density along distance from soma following exposure to 1 μM CDDP with or without 50 μM CX546 for 24 h. (F) Representative images and (G) quantification of PSD-95 puncta along distance from soma following 48 h co-treatment with 1 μM cisplatin and with or without 200 μM ampakine CX1739. (C) n = 9 neurons/group; (E, G) n = 6 neurons/group, 2 dendritic branches/neuron. Data are shown as mean ± SEM; not significant = ns, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined by two-way repeated measures ANOVA with Tukey’s post hoc analysis for multiple comparisons test. Scale bars = 10 μm.

Article Snippet: Ampakine CX546 (Tocris, 2980/10) was made into a 100 mM stock by dissolving in DMSO and stored at −20 °C.

Techniques: In Vitro, Expressing

Journal: Experimental neurology

Article Title: Cisplatin induces BDNF downregulation in middle-aged female rat model while BDNF enhancement attenuates cisplatin neurotoxicity

doi: 10.1016/j.expneurol.2024.114717

Figure Lengend Snippet: In vitro, screening of BDNF-enhancing pharmacological agents riluzole and CX546, and non-BDNF enhancing CX1739 in human ovarian cancer lines OVCAR8 and SKOV3.ip. OVCAR8 and SKOV3.ip1 were plated at 10,000 cells/well on 96-well plates and treated with 10 μM CDDP with or without graded doses of (A) BDNF (50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL), (B) riluzole (2.5 μM, 5 μM, 10 μM, 20 μM), (C) CX546 (50 μM, 100 μM, 200 μM, 300 μM), and (D) CX1739 (50 μM, 100 μM, 200 μM, 300 μM) for 72 h. Cell viability was normalized to vehicle control. (A-D) n = 3 wells/group. Data are shown as mean ± SEM; not significant = ns, *P < 0.05, **P < 0.01, ***P < 0.001, denotes statistical significance compared to vehicle control. ###P < 0.001 denotes statistical significance compared to 10 μM CDDP. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons test.

Article Snippet: Ampakine CX546 (Tocris, 2980/10) was made into a 100 mM stock by dissolving in DMSO and stored at −20 °C.

Techniques: In Vitro, Control

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with HPV genomes and the next day treated either with vehicle or different concentrations of CX4945 for 48 h. Total DNA was extracted from cells, digested with DpnI and restriction enzymes to linearize the HPV genomes, and analyzed using Southern blot (SB). B, C. U2OS cells were transfected with HPV18 genome, next day treated with different concentrations of CX4945 and incubated for the indicated periods of time. Cell cycle profile was analyzed using propidium iodide by flow cytometry. Viability of the cells was examined using MTT assay. D. U2OS cells were transfected with different HPV genomes and siRNAs simultaneously using electroporation. Treatment with 6 μM CX4945 was started 24 h post transfection. Levels of the linearized replicated HPV genomes were analyzed using SB. E. U2OS cells were transfected with HPV18 and siRNAs specific for CK2α and CK2α’ or scrambled Neg. siRNA simultaneously and incubated for 3 or 5 days. The cells incubated for 5 days, were transfected with the same siRNAs on the 3 rd day of incubation. Levels of CK2α, CK2α’ and tubulin proteins were detected using WB. F. left panel: U2OS cells were transfected with HPV18 genome, the next day treated with 6 μM CX4945, incubated for 3, 6, 9 and 12 days and analyzed using SB (lanes 1, 2, 3, 4, 7, 8, 9 and 10). One set of the cells treated with CX4945 for 6 days was switched to treatment with DMSO for additional 3 and 6 days that corresponded to 9 and 12 days post transfection (lanes 5 and 6). Right panel: SB signals from three independent experiments were quantified and set as 100% in the sample treated with DMSO for 3 days. Data are presented as an average mean +/- SD.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Southern Blot, Incubation, Flow Cytometry, MTT Assay, Electroporation

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with different HPVNLuc genomes, propagated for 2, 3 and 4 days and subjected to luciferase assay. Nluc activity was normalized to alkaline phosphatase activity. Normalized Nluc activity was set as 100% in the cells propagated for 2 days. B. U2OS cells were transfected with 250 or 400 ng of the HPV18NLuc genome and incubated for 3, 4 and 5 days. Total DNA was isolated and treated with DpnI and BglI. Replication of HPV18NLuc genome was analyzed using SB. C. U2OS cells were transfected with different amounts of HPV18NLuc genome and FFLuc encoding plasmid and incubated for the indicated periods of time. Signals of HPV18NLuc replication were quantified using ImageQuant software; the average means of three experiments +/- SD are shown (left panel). The NLuc activity was measured and normalized to FFLuc activity (right panel). Signals of HPV18NLuc replication or normalized NLuc activity were set as 1 in the sample transfected with 250 ng of HPV18NLuc and incubated for 3 days. D. U2OS cells were transfected with HPV18NLuc genome, FFLuc encoding plasmid and siRNAs if indicated. The next day, cells were treated with different concentrations of CX4945 or vehicle. Cells were incubated for 3, 4 or 5 days and subjected to luciferase assay. NLuc activity was normalized either to alkaline phosphatase activity in the samples treated with CX4945 or to FFLuc and alkaline phosphatase activities in the samples treated with siRNAs. Normalized NLuc activity in the cells treated with DMSO and incubated for 3 days was set as 1. E. U2OS cells were transfected with different amounts of HPV18NLuc genome and siRNAs. The next day, the cells were treated with 6 μM CX4945 or vehicle. Total DNA was extracted, treated with DpnI and BglI and analyzed using SB. F. NHEKs were transfected with different HPVNLuc genomes, incubated for 48 h and treated with 1 μM CX4945, if indicated. Nluc activity was normalized to total protein concentrations and set as 100% in the samples incubated for 2 days after transfection. NA–not analyzed. All panels: normalized NLuc data are presented as an average mean +/- SD of at least 3 replicates measured in 3 independent experiments.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Luciferase, Activity Assay, Incubation, Isolation, Plasmid Preparation, Software

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. CIN612 cells were transfected with siRNAs one or two times and propagated for 3 or 5 days. Levels of CK2α and CK2α’ proteins were detected using WB. B. CIN612 cells were transfected with siRNAs as is described in the panel A. Levels of CK2α and CK2α’ mRNA expression were measured using qPCR, normalized to GAPDH expression level and set as 100% in a sample transfected with Neg. siRNA. Data from other samples were calculated relative to that. Data are presented as the average mean +/- SD of three independent experiments performed in triplicate. C. CIN612 cells were subjected to single or double transfection with siRNAs and incubated for 3, 5 and 6 days, respectively. Total DNA was extracted, treated with HindIII restriction enzyme and subjected to SB analysis (left panel). SB signals obtained from three independent experiments were quantified, and data from the sample treated with Neg. siRNA and incubated for 3 days was set as 100% (right panel). D. CIN612 cells were treated with the indicated concentrations of CX4945 for 3 or 6 days. Total DNA was digested with HindIII restriction enzyme to linearize the HPV31b genome and subjected to SB analysis. E. Viability of CIN612 cells incubated in the presence of different concentrations of CX4945 for 3 or 6 days was tested using MTT assay. F. CIN612 cells were treated with 0.5 μM CX4945 for 3 or 6 days. The levels of the respective gene mRNA expression were measured by qPCR using 2 different pairs of primers, normalized with GAPDH mRNA expression levels and set as 1 in the samples treated with DMSO for 3 days. G. CIN612 cells were transfected with different HPVNLuc genomes, incubated for 2 days and treated with 0.5 0.5 μM CX4945 for additional 2 and 3 days. NLuc activity was measured, normalized to total protein concentrations and set as 100% in the samples incubated for 2 days. NA–not analyzed. The linearized HPV31b genome was analyzed using SB in the respectively treated samples (right panel). All panels: data are presented as an average mean +/- SD.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Expressing, Incubation, MTT Assay, Activity Assay

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with HPV18E1HA and HPV18wt genomes, the next day treated with 6 μM CX4945 or vehicle and propagated for 3, 4 and 5 days. Total DNA was extracted, digested with DpnI and BglI restriction enzymes and analyzed using SB. Low molecular weight bands correspond to DpnI-sensitive input DNA. B. U2OS cells were transfected with the HPV18E1HA genome and propagated for 2, 3, 5 and 6 days. CX4945 was added to cells 2 days after transfection. Cells were fractionated for isolation of total DNA and WCEs. Total DNA was treated with DpnI and BglI restriction enzymes and analyzed using SB. HA-tagged E1 protein was immunoprecipitated from WCEs and analyzed using WB. Tubulin was used as a loading control. C. U2OS cells were transfected with HPV18E1HA construct and siRNAs, if indicated, incubated for 3 days and treated with 6 μM CX4945 for 24 h. Cells were detached using trypsin-EDTA and fractionated for nuclear (Nuc) and cytoplasmic (Cyt) extracts. Nuclear and cytoplasmic HA-tagged E1 protein was immunoprecipitated using r-a-HA antibody and analyzed by immunoblotting using m-a-HA antibody. The nuclear and cytoplasmic markers lamin B and GAPDH were used. D. U2OS cells were transfected with the HPV18 genome, incubated for 2 days and treated with 6 μM CX4945 for 24 h (left panel). Alternatively, U2OS cells were cotransfected with the HPV18 genome and indicated siRNAs (right panel). Total RNA was extracted, treated with Turbo DNase and used for cDNA synthesis. Levels of E1 , E2 , E1^E4 and E8^E2 transcripts were measured using qPCR, normalized to GAPDH expression level and set as 100% in samples treated with DMSO or transfected with Neg. siRNA. Data are presented as the average means +/- SD of three independent experiments performed in triplicate.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Molecular Weight, Isolation, Immunoprecipitation, Control, Construct, Incubation, Western Blot, cDNA Synthesis, Expressing

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with the HPV18E1HA genome and incubated for 3 days. Cells were treated with 6 μM CX4945 and/or 10 μM MG132 for 6 h. HA-tagged E1 protein was immunoprecipitated from whole cell extracts (WCEs) using r-a-HA antibody and analyzed by immunoblotting using m-a-HA-HRP antibody (left panel). WB signals from three independent experiments were quantified. E1 level was set as 100% in the cells treated with DMSO (right panel). B. U2OS cells were transfected with the HPV18E1HA genome, incubated for 3 days and treated with 6 μM CX4945 (lanes 3–12) and 10 μM MG132 (lanes 7 and 8) for the indicated periods of time. Nuclear (Nuc) and cytoplasmic (Cyt) extracts were isolated. Nuclear and cytoplasmic HA-tagged E1 protein was immunoprecipitated using r-a-HA antibody and analyzed by immunoblotting using a-HA-HRP antibody. The nuclear and cytoplasmic markers lamin B and GAPDH were used (left panel). WB signals obtained from at least three independent experiments were quantified. The E1 protein level was set as 100% in the NE of cells treated with DMSO. All panels: data are presented as the average means +/- SD of at least three independent experiments (*—p<0.05; ***—p<0.001).

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Isolation

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

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 2. I3P boosts COX2 expression. (A) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 8 h. The cells were lysed, mRNA was extracted, and the expression of Ptgs2 was quantified by qPCR (n 5 3 from three independent experiments). (B) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 6 and 24 h. (C) COX2 was measured by Western blotting (n 5 4 from three independent experiments), and quantification by densitometry is shown. (D) BMDMs were treated with various concentrations (31.25 mM to 1000 mM) of I3P or control media for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. (E) COX2 was measured by Western blotting (n 5 5 from three independent experiments), and quantification by densitometry is shown. (FH) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 4, 6, or 8 h. The expression of Ptges (F), Pgis (G), and Tbxas1 (H) was measured by qPCR (n 5 3 from three independent experiments). All genes were normalized to rps18. (I) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. The concentration of PGs in both the cell lysates and supernatants was quantified by ELISA (n 5 2 from two independent experiments). Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed for (A), (C), and (E). A two-way ANOVA was performed for (F) and (G). The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test for one-way ANOVA or the Sidak test for two-way ANOVA. **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Western Blot, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

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

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 3. I3P inhibits COX2 activity. (A) Schematic showing the structures of indole-3-acetic acid, indomethacin, and I3P. (B) BMDMs were treated with indomethacin (50 or 100 mM), I3P (0.5 or 1 mM), or the relevant vehicle control (DMSO or media) for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h, and COX2 levels were measured by Western blotting (n 5 3 from three independent experiments). (C) Quantification by densitometry is shown. (D) Schematic depicting hypothesis of I3P inhibition of COX2. (E) BMDMs were treated with 1 mM I3P for 45 min, followed by addition of 5 mM AA or vehicle (DMSO) for a further 15 min, prior to stimulation with LPS (100 ng/ml) for 24 h. The concentration of PGs in the supernatants was quantified by ELISA (E) (n 5 5 from three independent experiments). (F) COX2 activity was measured after incubation of 1 mg recombinant COX2 with various concen- trations (31.25 mM to 1000 mM) of I3P or vehicle control (DMSO) (n 5 3 from three independent experiments). Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed for (C). A two-way ANOVA was performed for (E). The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test for one-way ANOVA or the Sidak test for two-way ANOVA. *p < 0.05, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Activity Assay, Control, Western Blot, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

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

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 4. The augmentation of COX2 expression by I3P is regulated by a PG feedback loop. (A) BMDMs were treated with 10 mM PGE2 for 1 h and subsequently with 1 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (B) COX2 was measured by Western blotting (n 5 3 from three inde- pendent experiments), and quantification by densitometry is shown. (C) BMDMs were treated with 5 mM of the EP2 antago- nist AH6809 for 1 h and subsequently with 1 mM I3P for 1 h fur- ther, prior to stimulation with LPS (100 ng/ml) for 24 h. (D) COX2 was measured by Western blotting (n 5 4 from three inde- pendent experiments), and quantification by densitometry is shown. (E) BMDMs were treated with 10 mM of the EP4 antago- nist GW 627368X for 30 min and subsequently with 0.5 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (F) COX2 was measured by Western blotting (n 5 4 from three independent experiments), and quantification by densitome- try is shown. (G) BMDMs were treated with 10 mM forskolin for 30 min and subsequently with 1 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (H) COX2 was mea- sured by Western blotting (n 5 4 from three independent experi- ments), and quantification by densitometry is shown. Densitometry analysis is presented as mean ± SD. A one-way ANOVA was per- formed. The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Western Blot

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

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 5. The augmentation of COX2 expression by I3P is partially dependent on AhR activation. (A) BMDMs from AhR1/1 and AhR/ mice were pretreated with 10 mM 3MC for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. (B) COX2 levels were measured by Western blotting (n 5 3 from one independent experiment), and quantification by densitometry is shown. (C) BMDMs from AhR1/1 and AhR/

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Activation Assay, Western Blot

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

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 6. I3P blocks trypanosome lysate-induced PG production and LPS-induced PG production in human macrophages. (AC) BMDMs were pre- treated with 1 mM I3P or control media for 30 min before they were treated with 100 ng/ml IFN-g and 25 or 100 mg/ml trypanosome lysate for 24 h. COX2 expression was measured by Western blotting (n 5 3 from three independent experiments) (A), and quantification by densitometry is shown (B). (C) PGs were measured by ELISA (n 5 3 from three independent experiments). (DF) Primary human macrophages were pretreated with I3P (1 mM, 0.5 mM, or 0.25 mM) or control media for 1 h before they were treated with LPS (100 ng/ml) for 24 h. COX2 expression was measured by Western blotting (n 5 4 from three independent experiments) (D), and quantification by densitometry is shown (E). (F) PGs were measured by ELISA (n 5 4 from three independent experiments). (G) Schematic representation of inhibition of COX2 by I3P. Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed. The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test. ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition