Journal: Frontiers in Oncology

Article Title: Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

doi: 10.3389/fonc.2019.00043

Figure Lengend Snippet: Simplified drawing showing the oxygenation gradient from the blood vessel to tumor cells. Circulating macrophages and endothelial cells lining the tumor vessel are exposed to the physiological oxygen concentrations in blood (~12 O 2 ) while oxygen levels within the tumor decline as the distance from the functional blood vessel increases.

Article Snippet: Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Functional Assay

Journal: Frontiers in Oncology

Article Title: Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

doi: 10.3389/fonc.2019.00043

Figure Lengend Snippet: Novel experimental in vitro setting to reproduce different oxygenation patterns between the tumor-vessel interface. (A) The device was performed by assembling an upper (a) and a lower (c) chamber with a PDMS membrane (b) between both chambers. The wells of the upper chamber where adjusted to fit to a conventional transwell insert. (B) Scheme of the novel in vitro experimental setting developed to study the interactions between tumor cells and macrophages simultaneously exposed to different oxygen levels mimicking the pathophysiological conditions in the solid tumor microenvironment. (C) The novel setting was able to produce an oxygen gradient as measured by an oxygen optical sensor.

Article Snippet: Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: In Vitro, Membrane

Journal: Frontiers in Oncology

Article Title: Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

doi: 10.3389/fonc.2019.00043

Figure Lengend Snippet: Macrophage migration through a permeable membrane in co-culture with mouse melanoma (B16F10), breast (E0771), or renal (RENCA) cancer cells exposed to different oxygen levels. (A) Representative bright field images showing stained macrophages which migrated to the lower side of the transwell membrane after 24 h in co-culture with melanoma, breast, or renal cancer cells cultured under normoxic conditions (N-N) or under a physiological gradient of oxygen (H-N). In the absence of tumor cells (right), macrophages did not migrate through the transwell membrane. Scale bar = 250 μm. (B) The number of macrophages (RAW 264.7) that migrated to the lower side of the membrane was significantly higher when B16F10 (left) or E0771 (center) were selectively exposed to hypoxia (H-N) in comparison to normoxia (N-N), while no differences were found between treatments in the migration of macrophages co-cultured with RENCA cells (right). Data were normalized to the control group (20% O 2 ).

Article Snippet: Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Migration, Membrane, Co-Culture Assay, Staining, Cell Culture, Comparison, Control

Journal: Frontiers in Oncology

Article Title: Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

doi: 10.3389/fonc.2019.00043

Figure Lengend Snippet: Assessment of macrophage polarity. (A) Representative graphs obtained during FACS analyses which depict the median fluorescence intensities (MFI) for M1 and M2 markers in macrophages co-cultured with melanoma (left), breast (center) and renal (right) cancer cells exposed to hypoxia (H-N) or normoxia (N-N). Hypoxia-treated melanoma and breast tumor cells reduced the M1-like polarization of macrophages (A) , as reflected by an increase in the M2/M1 ratio compared to that observed when both cell types were exposed to normoxia (N-N) No significant changes in the phenotype of macrophages were observed when co-cultured with renal cancer cells exposed to different oxygen levels (B) . The M2/M1 ratio was calculated as the ratio of the median fluorescence intensity (MFI) of CD206/CD86 surface markers.

Article Snippet: Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Fluorescence, Cell Culture

Journal: Frontiers in Oncology

Article Title: Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

doi: 10.3389/fonc.2019.00043

Figure Lengend Snippet: Proliferation of melanoma, breast, and renal cancer cells. Melanoma (upper) and breast (middle) cancer cells increased their proliferative rates when cultured under low levels of oxygen either in single culture (H) or in co-culture with normoxic macrophages (H-N) when compared to N and (N-N) treatment groups, respectively. The hypoxia-induced proliferation observed in single culture (H) was further enhanced when tumor cells were co-cultured with normoxic macrophages (H-N). Renal cancer cell proliferation remained similar among the different treatment groups (right).

Article Snippet: Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Cell Culture, Co-Culture Assay

Journal: Frontiers in Oncology

Article Title: Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

doi: 10.3389/fonc.2019.00043

Figure Lengend Snippet: Expression of macrophage recruitment-related genes in melanoma, breast, and cancer cells in response to hypoxia. PTGS2 (A) and VEGFA ( B , center) gene expression was upregulated in hypoxia-treated breast and melanoma cancer cells, respectively, in comparison to normoxia. (C) EMAPII gene expression was reduced by hypoxia in breast (upper) and melanoma (center) tumor cells, while remained invariable in renal cancer cells (lower).

Article Snippet: Mouse macrophages (RAW264.7), mouse melanoma (B16F10), and mouse renal adenocarcinoma (RENCA) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Gene Expression, Comparison

Journal: Molecules

Article Title: Antioxidant Potential of Myrciaria tenella Fruit Extracts: In Vitro and In Vivo Protection Against Oxidative Stress

doi: 10.3390/molecules31040602

Figure Lengend Snippet: In vitro assays performed on RAW 264.7 macrophage cells. ( a ) cytotoxic evaluation of M. tenella fruit extracts at concentrations of 50, 100, and 250 µg/mL. The y-axis represents the percentage of MTT reduction, and the x-axis corresponds to the different M. tenella extracts at the indicated concentrations. ( b ) Effects of extracts on cell viability following oxidative stress induced by H 2 O 2 stressor. The y-axis indicates the percentage of MTT reduction, and the x-axis represents the different extracts tested at 100 µg/mL. NC (negative control) corresponds to macrophages cultured in medium only, while PC (positive control) represents macrophages treated with the H 2 O 2 oxidative stressor. ( c ) Percentage of nitric oxide (NO) production after treatment with M. tenella extracts. The y-axis represents NO production (%), and the x-axis corresponds to treatments with M. tenella extracts at 100 µg/mL in combination with the LPS (1 µg/mL) stimulus. NC represents macrophages cultured in medium only, and PC represents macrophages treated with LPS, with PC set as 100% NO production. VA: aqueous unripe fruit extract; VE: hydroethanolic unripe fruit extract; MA: aqueous ripe fruit extract; ME: hydroethanolic ripe fruit extract. In panels ( b , c ), different letters (a–d) indicate statistically significant differences between extracts and NC compared with PC, according to Tukey’s test ( p < 0.0001). In panel ( a ), letters indicate statistically significant differences among extracts, as determined by Tukey’s test ( p < 0.01).

Article Snippet: Mouse embryonic fibroblasts (NIH/3T3, ATCC ® CRL-1658 TM ) and mouse macrophage cells (RAW 264.7, ATCC ® TIB-71 TM ) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS).

Techniques: In Vitro, Negative Control, Cell Culture, Positive Control

Journal: Marine Drugs

Article Title: Structural Characterization and Anti-Inflammatory Properties of an Alginate Extracted from the Brown Seaweed Ericaria amentacea

doi: 10.3390/md24010041

Figure Lengend Snippet: MTT assay for the assessment of in vitro cytotoxicity. ( a ) In vitro cytotoxicity of SA at various (from 0.05 to 0.4 mg/m/L) concentrations evaluated on RAW 264.7 murine macrophages. ( b ) In vitro cytotoxicity of SA at the same concentrations evaluated on human differentiated THP-1 M0 macrophages. The results are expressed as a percentage of viability with respect to the control (untreated cells) and are the mean ± SD of three experiments performed in five replicates. ANOVA was significant (i.e., p < 0.05) in ( a ), with ** p < 0.01 vs. NT in Tukey post-test.

Article Snippet: The murine RAW 264.7 cell line and the human THP-1 cell line were acquired from the American Type Culture Collection (LGC Standards srl, Milan, Italy).

Techniques: MTT Assay, In Vitro, Control

Journal: Marine Drugs

Article Title: Structural Characterization and Anti-Inflammatory Properties of an Alginate Extracted from the Brown Seaweed Ericaria amentacea

doi: 10.3390/md24010041

Figure Lengend Snippet: E. amentacea SA inhibition of gene expression in LPS-activated RAW 264.7 macrophages. Gene expression measured by qPCR analysis of IL-1β, IL-6, CXCL5, TNF-α, and COX-2, after incubation for 8 h ( a ) and 24 h ( b ), with or without LPS (100 ng/mL), and in the presence or absence of 0.2 and 0.4 mg/mL SA. Data are normalized on the GAPDH housekeeping gene and expressed as mRNA fold increase compared to control, untreated cells. Results are the mean ± SD of three experiments performed in duplicate. ANOVA was significant in each histogram ( p < 0.05), with * p < 0.05, and ** p < 0.01 vs. LPS in Tukey post-test.

Article Snippet: The murine RAW 264.7 cell line and the human THP-1 cell line were acquired from the American Type Culture Collection (LGC Standards srl, Milan, Italy).

Techniques: Inhibition, Gene Expression, Incubation, Control

Journal: Marine Drugs

Article Title: Structural Characterization and Anti-Inflammatory Properties of an Alginate Extracted from the Brown Seaweed Ericaria amentacea

doi: 10.3390/md24010041

Figure Lengend Snippet: NO-scavenging activity and iNOS gene expression in cellular assay. ( a , b ) Intracellular NO production measured by Griess assay in RAW 264.7 murine macrophages incubated for 8 h ( a ) and 24 h ( b ) with LPS (100 ng/mL) in the presence or absence of 0.2 and 0.4 mg/mL of SA. The results are expressed as a percentage of NO production, with respect to control, untreated cells, and are the mean ± SD of two assays performed in triplicate. ( c , d ) Gene expression measured by qPCR analysis of iNOS after RAW 264.7 cell incubation, for 8 h ( c ) and 24 h ( d ), with or without 100 ng/mL LPS and in the presence or absence of 0.2 and 0.4 mg/mL SA. Data are normalized on the GAPDH housekeeping gene and expressed as mRNA fold increases compared to control, untreated cells. Results are the mean ± SD of three experiments performed in duplicate. ANOVA was significant in each histogram ( p < 0.05), with ** p < 0.01 vs. LPS in Tukey post-test and ## p < 0.01 vs. negative control in Tukey post-test.

Article Snippet: The murine RAW 264.7 cell line and the human THP-1 cell line were acquired from the American Type Culture Collection (LGC Standards srl, Milan, Italy).

Techniques: Activity Assay, Gene Expression, Griess Assay, Incubation, Control, Negative Control

Journal: The FEBS journal

Article Title: Control of transferrin expression by β-amyloid through the CP2 transcription factor.

doi: 10.1111/j.1742-4658.2010.07801.x

Figure Lengend Snippet: Fig. 2. Transcription factor CP2 increases the endogenous transferrin mRNA level. (A) Total RNA from HEK293 cells transfected with plas- mids expressing FLAG-tagged CP2 and ⁄ or CP2 shRNA was analyzed by RT-PCR using transferrin- and GAPDH-specific primers. The level of GAPDH was used as a loading control. Representative images of agarose gels are shown (upper panels) and band intensity was measured. Normalized transferrin levels were calculated relative to GAPDH (bottom panel). (B) HEK293 cells were transiently transfected with CP2 shRNA. RNA was extracted, and RT-PCR analysis was performed as in (A). (C) Lysates from HEK293 cells transfected with increasing amounts of plasmids encoding CP2 DNA, transferrin promoter–luciferase or CP2 shRNA vectors were analyzed for luciferase activity. All data were normalized to b-galactosidase activity. Data are expressed as fold increases compared to the control (upper panel). The expression lev- els of proteins were assessed by immunoblotting (bottom panels). Expression of b-tubulin was included as a loading control. (D) HEK293 cells were co-transfected with transferrin promoter–luciferase and pCMV-b-galactosidase with/without CP2 shRNA. Luciferase activity was measured 48 h after transfection and normalized to b-galaosidase activity. The expression levels of proteins were assessed by immunoblot- ting using CP2 antibody. All data are representative of three independent experiments, and statistical significance was determined using Tukey’s post hoc test (**P < 0.01).

Article Snippet: Polyclonal antibodies against transferrin (sc-22597) and b-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Luciferase, Activity Assay, Western Blot

Journal: The FEBS journal

Article Title: Control of transferrin expression by β-amyloid through the CP2 transcription factor.

doi: 10.1111/j.1742-4658.2010.07801.x

Figure Lengend Snippet: Fig. 3. The transferrin proximal promoter region contains a CP2 response element. (A) Transferrin promoter constructs used in transactiva- tion studies. Various mutants of human transferrin promoters were used, and are indicated as a mutant 1, mutant 2 and mutant 3. The method used for mutation of transferrin promoters is described in Experimental procedures. (B) Each of the constructs shown in (A) and pCMV-b-galactosidase were transiently transfected into the HEK293 cells, together with increasing amounts of plasmids encoding CP2 DNA or CP2 shRNA vectors. At 48 h after transfection, cells were lysed and subjected to luciferase assays. Data were normalized against b-galac- tosidase activity and are expressed as the relative luciferase units compared to the control. The level of CP2 expression in each group of cells was confirmed by Western blotting, an example of which is shown (bottom panel). Expression of b-tubulin was used as a loading control. All data are representative of three independent experiments, and statistical significance was determined using Tukey’s post hoc test (**P < 0.01; n.s., not significant).

Article Snippet: Polyclonal antibodies against transferrin (sc-22597) and b-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Construct, Mutagenesis, Transfection, shRNA, Luciferase, Activity Assay, Control, Expressing, Western Blot

Journal: The FEBS journal

Article Title: Control of transferrin expression by β-amyloid through the CP2 transcription factor.

doi: 10.1111/j.1742-4658.2010.07801.x

Figure Lengend Snippet: Fig. 4. CP2 is able to bind to the proximal transferrin promoter con- sensus element in vitro and in vivo. (A) An oligonucleotide probe covering a 19 bp CP2-dependent enhancer DNA segment of the transferrin promoter was used for CP2 binding by EMSA. GST- fused recombinant CP2 was purified from E. coli using GST beads. The CP2–DNA complex migrated slowly and the amount increased in a concentration-dependent manner (lanes 4 and 5). The exis- tence of CP2 in this slow-migrating complex was verified by adding CP2 antibody, which caused a supershift (lane 6). Rabbit IgG and non-labeled probe were included as negative controls (lanes 7 and 8). N.S., non-specifically bound probe. (B) Chromatin from mouse hippocampal cell line HT22 was cross-linked with endogenous CP2. After precipitation with antibody against CP2 and rabbit IgG, the transferrin promoter regions containing the CP2 element were amplified by PCR from the precipitated DNA (upper panel). The intensity of the transferrin promoter band was quantified by densitometry (bottom panel).

Article Snippet: Polyclonal antibodies against transferrin (sc-22597) and b-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: In Vitro, In Vivo, Binding Assay, Recombinant, Concentration Assay, Labeling

Journal: The FEBS journal

Article Title: Control of transferrin expression by β-amyloid through the CP2 transcription factor.

doi: 10.1111/j.1742-4658.2010.07801.x

Figure Lengend Snippet: Fig. 5. Treatment with Ab modulates the transcriptional activity of CP2 by enhancing its binding affinity to transferrin promoter. (A,B) Trans- ferrin reporter vectors and pCMV-b-galactosidase were transiently transfected into HT22 cells, with or without CP2 shRNA vectors. Forty- eight hours after transfection, cells were exposed to various concentrations of Ab1–42 (50 or 100 nM) and Ab25–35 (5, 10 and 20 lM) for 12 h, then luciferase activity was measured (upper panel). Data were normalized using b-galactosidase activity, and are expressed as the relative luciferase units compared to the control. Protein levels were verified by Western blotting using antibodies against transferrin, CP2 and b-tubulin (as a loading control) (bottom panels). The band intensity was measured, and transferrin protein levels were normalized relative to b-tubulin. (C,D) After transfection and Ab treatment in HT22 cells as described above, cells were cross-linked with 1% formaldehyde and chromatin immunoprecipitations were performed using the CP2 antibody or rabbit IgG as a negative control. Binding of CP2 to the transferrin was detected by performing PCR using primers to the highly conserved transferrin promoter site. The ChIP experiments were performed several times, and representative gel images are shown. The equivalent of 1% of the chromatin used for each ChIP assay was also run on each gel (left panels). After ChIP, the band intensity was measured and the normalized expression level under each condition was calculated relative to the input level (right panels). All data are representative of three independent experiments, and statistical significance was determined using Tukey’s post hoc test (**P < 0.01; n.s., not significant).

Article Snippet: Polyclonal antibodies against transferrin (sc-22597) and b-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Activity Assay, Binding Assay, Transfection, shRNA, Luciferase, Control, Western Blot, Negative Control, Expressing

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: (2A) RAW 264.7 cells were co-incubated with NIH1 (left) and ATCC 17978 (right) isogenic wild-type strains and Δ gtr6 mutants. (2B) RAW 264.7 cells were co-incubated with ATCC 17978, the HUMC1:: gtr6 mutant strain with repaired gtr6 , or wild-type HUMC1. (2C) RAW 264.7 cells were co-incubated with ATCC 17978 wild type, Δ gtr6 , Δ gtr6 /pSC1a (the knockout mutant with a plasmid-borne functional copy) in the presence of complement-active serum, and Δ gtr6 /pSC1a in the presence of heat-inactivated serum. *p < 0.001. (2D) Gentamicin protection assay with RAW 264.7 cells and wild-type ATCC 17978 (black bars) or ATCC 17978 Δ gtr6 (white bars). Cytochalasin D was added as an inhibitor of phagocytosis. Total bacteria plated for CFUs and expressed as a proportion of initial bacterial inoculum. * = significant vs. bacteria-only group, ⸸ = significant vs. bacteria + RAW 264.7 cell group. *,⸸ = p < 0.01 (2E) RAW 264.7 cells were incubated with ATCC 17978, HUMC1, ATCC 17978 Δ gtr6 , and HUMC1:: gtr6 . Stained with Wright-Giemsa stain, total magnification is 1000x. Results are from two repeat experiments with duplicate samples in each. White arrows denote adherent or internalized bacteria.

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Incubation, Mutagenesis, Knock-Out, Plasmid Preparation, Functional Assay, Staining, Giemsa Stain

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: ( 4A ) 2.0×10 8 CFU of ATCC 1778 and HUMC1 had total capsule carbohydrate capsule extracted in parallel and total carbohydrate content measured via phenol-sulfuric acid colorimetry. ( 4B ) Incubation of macrophages and bacteria with purified capsule from gtr6 + (ATCC 17978, 15827) and gtr6 - (HUMC1, ATCC 17978 Δ gtr6 ) strains. Extract-free uptake was used as a control. * p < 0.0001 (4C) RAW 264.7 cells were pre-incubated with soluble mannan (0.5mg/mL), laminarin (0.5mg/mL), and dextran sulfate (0.1mg/mL) or an untreated control prior to co-incubation with ATCC 17978. *p < 0.0001. Two biological replicates for in vitro . Wide bars denote median, error bars denote IQR.

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Colorimetric Assay, Incubation, Purification, In Vitro

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: (5A) RAW 264.7 cells were pre-incubated with anti-Dectin-1, anti-CR3, anti-MR neutralizing monoclonal antibodies or an isotype control prior to co-incubation with ATCC 17978. *p < 0.0005, * *p < 0.0001 (5B) Knockdown of Dectin-1 and/or CR3 in RAW 264.7 cells followed by incubation with ATCC 17978. *p < 0.0001 (5C) Primary peritoneally-elicited macrophages from C57BL/6 mice followed by phagocytosis assays with ATCC 17978. *p < 0.05, * *p < 0.0001 (5D) Phagocytosis assays of ATCC 17978 with peritoneal neutrophils from wild-type mice with disruption of phagocytosis upon the addition of heat-inactivated serum (HI-S) or complement-active serum (CA-S). *p < 0.0001 (5E) Phagocytosis assays with RAW 264.7 macrophages with gtr6 + and capsule-free strains (ATCC 17978 WT, 15827, ATCC 17978 ΔitrA ), and gtr6 - strains (ATCC 17978 Δgtr6 , HUMC1), with complement active (CA-S) or heat-inactivated (HI-S) serum. *p < 0.0001. Experiments repeated once with two biological replicates. Wide bars denote median, error bars denote IQR.

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Incubation

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: (6A) Incubation of RAW 264.7 cells with ATCC 17978 in the presence of 100μg/mL GlcNAc (NAG), a CR3 lectin domain inhibitor. (6B) Serial two-fold dilutions of complement-active mouse serum in a RAW 264.7 cell phagocytosis assay with ATCC 17978. *p < 0.0001 (6C) Male C57BL/6 mice aged 10 weeks were infected intravenously with 2.0×10 8 CFUs of 15827, with or without administration of 15μg cobra venom factor (CVF) 48 h prior to infection. *p < 0.001. Experiments repeated once, n = 5 per group for in vivo and two technical replicates for in vitro .

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Incubation, Phagocytosis Assay, Infection, Combined Bisulfite Restriction Analysis Assay, In Vivo, In Vitro

Journal:

Article Title: Papillomavirus Capsid Mutation To Escape Dendritic Cell-Dependent Innate Immunity in Cervical Cancer

doi: 10.1128/JVI.79.11.6741-6750.2005

Figure Lengend Snippet: VLP, but not assembly-defective HPV16 L1, mutants activate NF-κB- and AP-1-dependent transcription via MyD88. (A) The influence of in vitro-assembled HPV16 L1 mutant preparations upon NF-κB- and AP-1-dependent transcription in the macrophage cell line RAW264.7. Fold increase in chemiluminescence intensity after stimulation compared to unstimulated cells is plotted. (B) Assessment of the mRNA level of MyD88 in RAW264.7 cells by RT-PCR after stable transfection with siRNA constructs targeting MyD88 mRNA. (C) MyD88 knockdown (KD) blunts the NF-κB-dependent transcriptional response to TLR ligands LPS and CpG as well as HPV16 VLP. Results from three experiments are shown.

Article Snippet: The RAW264.7 MyD88 knockdown cell lines were maintained in Dulbecco's modified Eagle medium-10% FCS-hygromycin B. Transfection and reporter assays. pNF-κB-SEAP, pAP1-SEAP, and positive-control pSEAP2 as well as negative-control pTAL-SEAP reporter constructs (Clontech) were individually transfected into mouse RAW-264.7 macrophage cells with Lipofectamine 2000 (Invitrogen) in 24-well plates.

Techniques: In Vitro, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Construct

Journal:

Article Title: Papillomavirus Capsid Mutation To Escape Dendritic Cell-Dependent Innate Immunity in Cervical Cancer

doi: 10.1128/JVI.79.11.6741-6750.2005

Figure Lengend Snippet: Mechanisms driving the induction of HPV16 L1 VLP-specific IgG1 and IgG2a. (A) ELISA of HPV16 L1 VLP-specific IgG1 and IgG2a titers in the pooled sera of C57BL/6 mice (n = 6) immunized with in vitro-assembled preparations of each HPV16 L1 mutant. (B) Production of HPV16 L1 VLP-specific IgG1 and IgG2a in MyD88-deficient and control mice. (C) Production of HPV16 L1 VLP-specific IgG1 and IgG2a in IFN-α/β receptor-deficient and control mice. (D) Production of HPV16 L1 VLP-specific IgG1 and IgG2a in CD4-deficient and control mice. The mice were immunized with HPV16 L1 VLP on day 0, day 7, and day 14. The sera were collected at 10 days after final vaccination and analyzed using a mouse isotyping kit. ELISA plates were coated with 100 ng/well of the immunogen, and representative results from three experiments are shown.

Article Snippet: The RAW264.7 MyD88 knockdown cell lines were maintained in Dulbecco's modified Eagle medium-10% FCS-hygromycin B. Transfection and reporter assays. pNF-κB-SEAP, pAP1-SEAP, and positive-control pSEAP2 as well as negative-control pTAL-SEAP reporter constructs (Clontech) were individually transfected into mouse RAW-264.7 macrophage cells with Lipofectamine 2000 (Invitrogen) in 24-well plates.

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Mutagenesis