Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 and NF- κ B signalling pathways are deregulated in human CC, with ERK5 expression correlating with increased NF- κ B activation. ( a ) Representative immunoblots of steady-state protein expression levels of MEK5, ERK5, NF- κ B, I κ B and β -actin in normal colon, colon adenomas and pMMR and dMMR colon carcinomas; ( b ) MEK5 and ERK5 steady-state protein levels; ( c ) NF- κ B, I κ B and NF- κ B/I κ B ratio; ( d ) correlations between ERK5 steady-state protein levels and NF- κ B, I κ B or NF- κ B/I κ B ratio; ( e ) representative immunoblots of steady-state levels of p-NF- κ B, NF- κ B, p-I κ B, I κ B and β -actin in normal colon, colon adenoma, and pMMR and dMMR colon carcinomas; and ( f ) representative immunohistochemistry for ERK5, p-NF- κ B and NF- κ B in human colon cancer samples. Immunoblot statistical significance was determined using the non-parametric statistical analysis Kruskal–Wallis test with Dunn's post test for selected comparisons and results are expressed as mean±S.E.M. for samples in each category; correlation statistical significance was determined using the non-parametric stastistical analysis Spearman test. pMMR, proficient mismatch repair system; dMMR, deficient mismatch repair system. Scale bar=100 μ m. * P <0.05, † P <0.01 and ‡ P <0.001 from normal colon

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Expressing, Activation Assay, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: Correlation between MEK5, ERK5, NF- κ B and I κ B steady-state protein expression, NF- κ B activation and clinicopathological characteristics

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Expressing, Activation Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation accelerates cell cycle progression in SW620 cells. ( a ) Representative immunoblot of ERK5 protein levels in SW620 cell lines with differential ERK5 activation, stable constitutive MEK5 activation ( CA-MEK5 ), dominant-negative MEK5 ( DN-MEK5 ), empty control ( Empty ) and wild-type SW620 cell line (WT). The developed cell model consistently showed that DN-MEK5 led to constitutive inhibition of ERK5 activation, CA-MEK5 led to constitutive ERK5 activation and Empty control cells displayed basal ERK5 activation. ( b ) FACS cell cycle analysis of CA-MEK5, DN-MEK5 and Empty SW620 cells, following release from dual-thymidine block and exposed to ( c ) XMD8-92 or ( d ) BAY11-7085. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Western Blot, Dominant Negative Mutation, Control, Inhibition, Cell Cycle Assay, Blocking Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation accelerated cell cycle is associated with NF- κ B activation via reduction of I κ B steady-state levels. Immunoblot analysis of steady-state protein levels of ( a ) p-ERK5, ERK5 and p-ERK5/ERK5, and of ( b ) p-NF- κ B/NF- κ B, NF- κ B/I κ B ratios, p-I κ B and I κ B. ( c ) Representative immunoblots for p-ERK5, ERK5, p-NF- κ B, NF- κ B, p-I κ B, I κ B and β -actin in Empty, DN-MEK5 and CA-MEK5 SW620 cells when treated for 24 h with XMD8-92 and BAY11-7085, or untreated (no addition). Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05, † P <0.01 and ‡ P <0.001 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increases cell migration in vitro . Cell migration was assessed by ( a ) wound-healing assay at 24 and 48 h after ‘wound' formation and ( b ) transwell migration assay, with cells allowed to migrate for 9 h after cell platting. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Migration, In Vitro, Wound Healing Assay, Transwell Migration Assay

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increased cell migration is associated with increased vimentin expression and NF- κ B activation. Immunoblot analysis of steady-state protein levels of ( a ) vimentin and ( b ) of p-NF- κ B/NF- κ B, NF- κ B/I κ B ratios, p-I κ B and I κ B. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. of at least three independent experiments. * P <0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Migration, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: MEK5/ERK5 activation increases NF- κ B nuclear translocation and transcriptional activity via I κ B phosphorylation and degradation. Immunoblot analysis of steady-state protein levels of (a) p-I κ B, I κ B and p-I κ B/I κ B ratios, and ( b ) nuclear and cytosolic NF- κ B; ( c ) NF- κ B transcriptional activity, evaluated by dual luciferase assay with reporter plasmids. Significance was determined using ANOVA test with Tukey's post test for selected comparisons and results are expressed as mean±S.E.M. from at least three independent experiments. * P< 0.05 from Empty cell line

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: Activation Assay, Translocation Assay, Activity Assay, Phospho-proteomics, Western Blot, Luciferase

Journal: Cell Death & Disease

Article Title: Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF- κ B activation

doi: 10.1038/cddis.2015.83

Figure Lengend Snippet: In vivo , MEK5/ERK5 activation is associated with local invasion and regional lymphnode metastasis. As shown in the graphical abstract, ( a ) the injected tumours may grow locally in the caecum/colon infiltrating mucosa, sub-mucosa, muscularis propria and eventually sub-serosa. In addition, tumour growth may be focal (restricted to one foci, at the injection site) or multifocal (numerous foci spread throughout the caecum and colon, not restricted to the injection site). Furthermore, in addition to local invasion by tumour cells, the metastatic cascade encompasses intravasation into lymph vessels, extravasation out of the circulation, and survival and growth at secondary site. We injected 5 × 10 5 SW620 DN-MEK5 or CA-MEK5 cells into the wall of the caecum, in BALB/c scid mice, and mice were killed 30 or 60 days post injection. ( a ) Histopathological characteristics of DN-MEK5 and CA-MEK5 tumours regarding local invasion, extravasation and distant metastasis (to regional lymphnodes), and ( b) representative microphotographs of the multifocallity of CA-MEK5 tumours, compared with the focal lesions seen in DN-MEK5 tumours (white arrows, upper panel), of the lymph vessel invasion (black arrowhead, middle panel) and of the lymphnode metastasis (lower panel). *Tumour cells

Article Snippet: In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF- κ B inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μ M, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Techniques: In Vivo, Activation Assay, Injection

Journal: Burns & Trauma

Article Title: Pterostilbene accelerates wound healing by modulating diabetes-induced estrogen receptor β suppression in hematopoietic stem cells

doi: 10.1093/burnst/tkaa045

Figure Lengend Snippet: BMT of PTE-treated diabetic HSCs protects against diabetes-induced oxidative stress in PBMCs. Experimental rats were randomly separated into 4 groups as follows: rats with BMT of HSCs from CTL/VEH (BMT-CTL/VEH); rats with BMT of HSCs from STZ/VEH (BMT-STZ/VEH); rats with BMT of HSCs from STZ/RSV (BMT-STZ/RSV); and rats with BMT of HSCs from STZ/PTE (BMT-STZ/PTE). The rats were subjected to a model of cutaneous burn injury and PBMCs were collected for biomedical analysis after a 3-week period post-burn. (a) ROS formation in PBMCs, n = 5. (b) Quantitation of 3-nitrotyrosine formation, n = 5. (c) 8-OHdG formation, n = 5. (d) Quantitation of γH2AX formation. (e) Representative γH2AX western blotting band for (d), n = 5. (f) SOD2 activity, n = 5. (g) Quantitation of 8-oxo-dG formation, n = 5. (h) Representative pictures of 8-oxo-dG staining for oxidative stress (green) and DAPI staining for nuclei (blue) in PBMC, n = 4. For bars in graphs marked with an asterisk, p < 0.05 vs BMT-CTL/VEH group. Data are expressed as mean ± SEM. BMT bone marrow transplantation, PTE pterostilbene, HSCs hematopoietic stem cells, PBMCs peripheral blood mononuclear cells, CTL control, VEH vehicle, STZ streptozotocin, RSV resveratrol, ROS reactive oxygen species, OHdG 8-hydroxy-2′-deoxyguanosine, γH2AX phospho-Ser139 histone H2A.X, SOD2 superoxide dismutase 2, DAPI 4,6-diamidino-2-phenylindole, H2AX H2A.X Variant Histone, 8-oxo-dG 8-Oxo-2′-deoxyguanosine

Article Snippet: After blocking with 5% goat serum in PBS at room temperature for 30 minutes, cells were incubated with 8-oxo-dG Anti-mouse antibody (# 4354-MC-050, from Novus Biologicals) for 12 hours at 4°C and subsequently with secondary antibody Alexa Fluor 488.

Techniques: Quantitation Assay, Western Blot, Activity Assay, Staining, Transplantation Assay, Control, Variant Assay

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: Establishment of basal cell (BC) and endothelial cell (EC) co-culture system to study BC differentiation into a mucociliated epithelium. A. Immunofluorescence assessment of a normal human airway biopsy with staining for BC (KRT5, green), EC (CD31, red) and nuclei (DAPI, blue). Scale bar 50 μm. B. Schematic representation of the BC and EC co-culture system to study the impact of EC on BC differentiation into a mucociliated epithelium. C. Immunofluorescence assessment of BC and EC co-culture with staining for BC (KRT5, red), EC (VE-cadherin, green) and nuclei (DAPI, blue). Upper panel, BC cultured alone. Lower panel, BC and EC co-culture. White dashed line outlines the membrane of the Transwell insert. Scale bar 20 μm. D. Immunofluorescence staining of the epithelial layer of ALI day 28 cultures. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio and then stained for ciliated cells (β-tubulin IV, green), secretory cells (SCGB1A1, red) and nuclei (DAPI, blue). Scale bar 20 μm. E. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 7 days to assess the impact of EC on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell (FOXJ1) markers in BC cultured alone or co-cultured with EC cells at 10:1 and 2:1 (BC:EC) ratio. Bars indicate the mean for n=6 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC alone. The experiments for E were performed with n=4 independent donors of BC and EC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Cell Culture, Membrane, Expressing

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: Endothelial cell (EC) co-culture increases differentiation of basal cells (BC) into ciliated cells. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 28 days to assess the impact of EC on BC differentiation into a mucociliated epithelium at the histological level via immunofluorescence staining with cell type specific markers. A. KRT5+ BC. Sections of cells were stained for KRT5 (red) and DAPI (nuclei, blue). B. KRT8+ intermediate cells. Sections of cells were stained for KRT8 (red) and DAPI (nuclei, blue). C. SCGB1A1+ secretory cells. Sections of cells were stained for SCGB1A1 (red) and DAPI (nuclei, blue). D. MUC5B+ secretory cells. Sections of cells were stained for MUC5B (red) and DAPI (nuclei, blue). E β-tubulin IV+ cells. Sections of cells were stained for β-tubulin IV (ciliated, green) and DAPI (nuclei, blue). A–E. Scale bar 20 μm. H. Quantification of KRT5+, KRT8+, SCGB1A1+, MUC5B+ and β-tubulin IV+ cells. The bars indicate the mean for n=4 independent experiments and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC cultured alone. The experiments for A–H were performed with n=4 independent donors of BC and EC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Staining

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: siRNA mediated knockdown of INSR and IGF1R in basal cells (BC) suppresses ciliated cell differentiation. Basal cells with transfected with either control, INSR or IGF1R specific siRNA and cultured on ALI for 7 days to assess the impact of each receptor on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. A. TaqMan PCR analysis to assess INSR mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. B. TaqMan PCR analysis to assess IGF1R mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. C. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell markers (FOXJ1, MYB and DNAI1) in BC at ALI day 7 following siRNA mediated knockdown of INSR (black bars) and IGF1R (grey bars). Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC transfected with control siRNA. The experiments for A–C were performed with n=3 independent donors of BC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Knockdown, Cell Differentiation, Transfection, Control, Cell Culture, Expressing

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 1. Initial steps of aerobic cholesterol degradation in bacteria. Sterol degradation proceeds via steroid ring oxidation and side chain degradation (upper route). The exact order of side chain degradation and ring oxidation in vivo is unknown. The depicted metabolites are: (1) cholest-5-en-3-ol (choles- terol), (1) cholest-4-en-3-one, (2) 26OHCh, (2) 26OHCh-3O, (3) 3OHChA, (3) 3OChA, (4 and 4) the corresponding CoA derivatives of 3 and 3, (5) AD, and (6) ADD. CYP125 and CYP142, steroid 26-monoxigenases; 3--HSD, 3--hydroxysteroid dehydrogenase; KstD, 3-ketosteroid 1-dehydrogenase; FadD, steroid CoA ligase.

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: Bacteria, In Vivo

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 2. Interaction of KstR with palmitic acid, 3OHChA, and 3OChA induce a conformational change that destabilizes the protein. KstR thermal denaturation profiles were measured by circular dichroism in the presence of increasing concentrations of palmitic acid (A), 3OHChA (B), 3OChA (C), and 50 M of sodium cholate, sodium deoxycholate, and palmitic acid (D).

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: Circular Dichroism

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 3. Effect of palmitic acid, 3OHChA, and 3OChA on the far-UV CD spectra of KstR. The addition of palmitic acid (red line), 3OHChA (green line), and 3OChA (blue line) induced a reduction in the amount of helical content of KstR (black line), as indicated by diminished intensity of the spectra in the presence of these compounds. The effect was greater with the addition of saturating concentrations of 3OChA or 3OHChA followed by a smaller effect for palmitic acid.

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: Circular Dichroism

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 4. Sedimentation velocity profiles of KstR. The assay was performed in the absence (A) and presence of palmitic acid (B), 3OHChA (C), and 3OChA (D). The distribution pattern of concentration of the protein (c(s)) is represented in front of the sedimentation coefficient (S). The protein concentration used was 40 M. There are no differences in the oligomerization state of KstR when ligands are present, and most of the protein was present as a dimer in solution.

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: Sedimentation, Concentration Assay, Protein Concentration

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 5. Inhibition of KstR-P5228 interaction by 3OChA. A, analysis by EMSA of KstR binding to the P5228 promoter. The 5228FP probe concentration was 0.5 nM. Purified KstR were used at 0 nM (lane 1), 100 nM (lane 2), 500 nM (lane 3), and 1 M (lane 4). For lanes 5–10, the KstR concentration was 500 nM. Lanes 5–7 contained unlabeled 5228FP DNA probe (40-, 400-, and 4000-fold, respectively). Lanes 8–10 contained unrelated DNA from salmon sperm (0.5, 1.0, and 2.0 g, respectively). B, analysis by EMSA of the KstR-5228FP DNA probe complex in the presence of 3OChA, palmitic acid, and 3OHChA. KstR concentration was 500 nM, except in lanes 1 and 16 (0 nM). 3OChA (lanes 4–9), palmitic acid (lanes 10–15), and 3OHChA (lanes 19–24) were added at 1, 3, 6, 12, 25, and 50 M. KstR plus 5% of methanol (lane 3 and 18) was included as control. C, analysis by EMSA of the KstR-5228FP complex in the presence of related compounds at 250 M: cholestenone (lane 4), cholesterol (lane 5), 3OChA (lane 6), 3OHChA (lane 7), ADD (lane 8), palmitic acid (lane 9), palmitoyl alcohol(lane10),oleicacid(lane11),oleylalcohol(lane12),1-octanol(lane13), and octanoic acid (lane 14). Controls without inducers (lane 2) and with 5% of methanol were included. KstR concentration was 500 nM except in lane 1 (0 nM). D, determination of the apparent Kd for KstR binding to the 5228FP probe in the presence of 3OChA. The apparent Kd is the 3OChA concentration at which 50% of the total DNA probe was bound to KstR. This value was deter- mined from the experiment in B.

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: Inhibition, Binding Assay, Concentration Assay, Purification, Control

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 6. KstR binding to the chimeric promoter PQ3 in the presence of 3OChA and palmitic acid. A, sequence of P5228, PlacUV5, and PQ3. The operator sequence for KstR at P5228 and PQ3 promoters is boxed in dark gray. The 35 and 10 boxes, the ribosome binding sites (RBS), and the transcription start site (1) are indicated. LacI operator sequence at PlacUV5 and PQ3 is boxed in light gray. The direction of transcription is indicated by an arrow. B, analysis by EMSA of KstR binding to the PQ3 promoter. PQ3 probe concentration was 0.5 nM. The concentrations of purified KstR used were 0 nM (lanes 1 and 6), 100 nM (lane 2), 250 nM (lane 3), 500 nM (lane 4), 1 M (lane 5), and 500 nM (lanes 7–13). Lanes 8–10 contain unlabeled PQ3 DNA probe (40-, 400-, and 4000-fold, respectively), and lanes 11–13 contain unrelated DNA from salmon sperm (0.5, 1.0, and 2.0 g, respectively). C, analysis by EMSA of the KstR-PQ3 DNA probe complex in the presence of 3OChA and palmitic acid. KstR concentration was 500 nM, except in lane 1 (0 nM). 3OChA (lanes 4–9) was added at 1, 3, 6, 12, 25, and 50 M. Palmitic acid(lanes10–15)wasaddedat10,50,100,250,and500Mand1mM.KstRcontaining5%ofmethanol(lane3)wasalsoincludedascontrol.D,analysisbyEMSA of the KstR-PQ3 DNA probe complex in the presence of 3OHChA. KstR concentration was 500 nM, except in lane 1 (0 nM). 3OHChA (lanes 4–9) was added at 10, 50, 100, 250, and 500 M and 1 mM. KstR containing 5% methanol (lane 3) was also included as control. E, determination of apparent Kd for KstR binding to the PQ3 DNA probe in the presence of 3OChA and palmitic acid. The apparent Kd is the compound concentration at which 50% of the total probe was bound to KstR. This value was determined from the experiment in C.

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: Binding Assay, Sequencing, Concentration Assay, Purification, Control

Journal: Journal of Biological Chemistry

Article Title: Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

doi: 10.1074/jbc.m113.545715

Figure Lengend Snippet: FIGURE 8. 3OChA induces transcription from KstR-repressed promoters in vitro. Multiple-round in vitro transcription was performed in the presence of 40 nM of RNAP and 500 nM of KstR. and indicate the presence and absence, respectively, of KstR and methanol in each reaction. PQ3-derived mRNA (152 nucleotides) and the vector-derived mRNA control (RNAI, 108–109 nucleotides) are indicated. A, multiple-round in vitro transcription in the presence of increasing concentrations of 3OChA. Reactions were carried out with the plasmid pJCD01 as a control (lane 1) and plasmid pJCDPQ3 containing the PQ3 promoter (lanes 2–11). 3OChA was added at 1, 3, 6, 12, 25, and 50 M (lanes 6–11). B, multiple-round in vitro transcription in the presence of 250 M of cholesterol, cholestenone, 3OHChA, 3OChA, and palmitic acid (lanes 5–9). Reactions were performed with the plasmid pJCD01 (lane 1) and pJCDPQ3 (lanes 2–9). The picture is a representative example of four experiments. C, PQ3 transcription levels from pJCDPQ3 in the presence of KstR and inducers. The intensity of the bands observed in the in vitro transcription experiments (Fig. 8B) was used to calculated the transcription percentages, taking into account the intensity of the band in the absence of KstR (Fig. 8B, lane 2) as 100% of the transcription. Bars represent averages of four independent experiments, and errors bars indicate S.D. OL, cholesterol; ONE, cholestenone; PA, palmitic acid.

Article Snippet: Choles-5-ene-3 ,26-diol (26-hydroxycholesterol (26OHCh)), 3OHChA, and 3OChA were obtained from Avanti Polar Lipids.

Techniques: In Vitro, Derivative Assay, Plasmid Preparation, Control

Journal:

Article Title: Selective Inhibition of the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein by 1,25(OH) 2 Vitamin D 3

doi: 10.1016/j.molimm.2005.07.023

Figure Lengend Snippet: Effect of 1,25(OH)2D3 on chemotaxis to C5a, LTB4, formyl peptide and CXCL8. Purified DBP (100 nM) was treated for 5 min at 37°C with 10 nM 1,25(OH)2D3. Neutrophils (4 x 106/ml) were then preincubated for 40 min at 37°C with either DBP plus 1,25(OH)2D3 or buffer. Neutrophil movement then was assayed for 30 min at 37°C. Numbers represent mean ± SEM of 3 experiments using cells from different donors.

Article Snippet: Recombinant CXCL8 (IL-8) was obtained from R&D Systems (Minneapolis, MN).

Techniques: Chemotaxis Assay, Purification