Journal: Biochemical pharmacology

Article Title: Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells.

doi: 10.1016/j.bcp.2009.06.003

Figure Lengend Snippet: Fig. 2. Changes in receptor expression and intracellular Ca2+ in THP-1 cells. (A) Flow cytometry analysis of HEK.CCR5 and THP-1 cells after MCD and lovastatin treatment, cells were treated with lovastatin for 3 days and MCD for 1 h, respectively, stained with an anti-CCR5 antibody and the corresponding FITC-conjugated secondary antibody and analysed using flow cytometry, data represents percentage of Geo Mean fluorescence compared to vehicle treated (control) cells. (B) Flow cytometry analysis of CCR5 expression on control THP-1 cells and cells after MCD treatment, respectively. MCD treatment increases the expression level of CCR5. (C) THP-1 cells were treated with 10 mM MCD as described and when indicated 10 mM cholesterol was fed back to the cells before activation of receptor with 100 nM CCL3. (D) Single calcium trace of THP-1 cells either pre-treated with 10 mM MCD, 10 mM cholesterol or 10 mM MCD plus 10 mM cholesterol before receptor activation with 100 nM CCL3 when indicated. (E) Cells were treated with PTX or vehicle overnight and MCD was added 1 h before calcium flux was induced with 100 nM CCL3. (F) Single calcium trace of THP-1 cells treated with PTX overnight and/or MCD for 1 h before addition of 100 nM CCL3. Significant changes towards control cells are indicated by asterisks (*p < 0.05, **p < 0.01). Data are expressed as either changes in fluorescence ratio [340 nm/380 nm], where the basal line before addition of chemokine is subtracted from the peak fluorescence after addition of chemokine or as percentage of stimulation over basal, where the basal level is normalised to 100%. Data represent mean s.e.m. from at least three independent experiments for the bar charts and a representative tracer for the calcium flux.

Article Snippet: Lovastatin was obtained from Tocris (Bristol, UK) and activated before use as described by the manufacturer.

Techniques: Expressing, Flow Cytometry, Staining, Cytometry, Control, Activation Assay

Journal: Biochemical pharmacology

Article Title: Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells.

doi: 10.1016/j.bcp.2009.06.003

Figure Lengend Snippet: Fig. 3. Effect of statins on release of intracellular calcium in THP-1 cells. Cells were incubated with different statins for 3 days before cells were stimulated with 100 nM CCL3. (A) THP-1 cells pre-treated with lovastatin for 3 days, MCD for 1 h or vehicle (control) before receptor was activated with 100 nM CCL3, and single trace of THP-1 cells pre- treated with vehicle (control), MCD, lovastatin or MCD and lovastatin before the addition of 100 nM CCL3 as indicated. (B) The change of intracellular calcium in THP-1 cells, pre-treated with different concentrations of simvastatin for 3 days or vehicle (control) and challenged with 100 nM CCL3 and single trace of THP-1 cells pre-treated with vehicle (control) or simvastatin before the addition of 100 nM CCL3 when indicated. Significant changes to control data are shown by asterisks (**p < 0.01), Data are expressed as either changes in fluorescence ratio [340 nm/380 nm], where the basal line before addition of chemokine is subtracted from the peak fluorescence after addition of chemokine or as percentage of stimulation over basal, where the basal level is normalised to 100%. Data represent mean s.e.m. from at least three independent experiments for the bar charts and a representative tracer for the calcium flux.

Article Snippet: Lovastatin was obtained from Tocris (Bristol, UK) and activated before use as described by the manufacturer.

Techniques: Incubation, Control

Journal: Biochemical pharmacology

Article Title: Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells.

doi: 10.1016/j.bcp.2009.06.003

Figure Lengend Snippet: Fig. 4. Effect of statins on release of intracellular calcium in HEK.CCR5 and CHO.CCR5 cells. (A) HEK.CCR5 cells pre-treated with vehicle (control), lovastatin for 3 days, MCD for 1 h, or both before the receptor is activated with 100 nM CCL3. Significant changes to control data are shown by asterisks (*p < 0.05, **p < 0.01). (B) CHO.CCR5 cells pre- treated with vehicle (control), lovastatin for 3 days, MCD for 1 h, or both before the receptor is activated with 100 nM CCL3. Data are expressed as either changes in fluorescence ratio [340 nm/380 nm], where the basal line before addition of chemokine is subtracted from the peak fluorescence after addition of chemokine or as percentage of stimulation over basal, where the basal level is normalised to 100%. Data represent mean s.e.m. from at least three independent experiments for the bar charts and a representative tracer for the calcium flux.

Article Snippet: Lovastatin was obtained from Tocris (Bristol, UK) and activated before use as described by the manufacturer.

Techniques: Control

Journal: Biochemical pharmacology

Article Title: Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells.

doi: 10.1016/j.bcp.2009.06.003

Figure Lengend Snippet: Fig. 5. Quantification of cholesterol levels in the plasma membrane of different cells. (A) THP-1 cells were incubated with different concentrations of lovastatin for 3 days and cholesterol levels in the cells were determined using the Amplex Red cholesterol assay as described. Increasing concentrations of lovastatin decrease the amount of cholesterol present in the cells. (B) THP-1 cells were treated with different concentrations of simvastatin for 3 days. (C) THP-1 cells were treated with MCD and filipin for 1 h. (D) HEK.CCR5 cells were treated with either MCD or filipin for 1 h or lovastatin for 3 days. Data are represented as percentage of cholesterol content compared to vehicle treated (control) cells. Significant changes towards control are shown by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Data represent mean s.e.m. of at least three independent experiments.

Article Snippet: Lovastatin was obtained from Tocris (Bristol, UK) and activated before use as described by the manufacturer.

Techniques: Clinical Proteomics, Membrane, Incubation, Amplex Red Cholesterol Assay, Control

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Cell proliferation of human breast cancer cell lines . (a) MDAMB231 and (b) MDAMB468. Cells were treated with increasing concentrations of lovastatin lactone or lovastatin acid (μg/mL) for 48 hours. Data are presented as mean ± standard deviation (n = 5) * P < 0.05;** P < 0.05; *** P < 0.001.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Changes in expression of proteins involved in (a) regulation of cell cycle and cell death and (b) oxidative and metabolic processes of human MDAMB231 and MDAMB468 cells . Both cell lines were treated with 8 μg/mL lovastatin lactone (Lova Lac) or lovastatin acid (Lova Ac) for 48 hours. Data represent relative spot volumes (as calculated from two-dimensional gel images of whole cell extracts; data are presented as mean ± standard deviation (n = 5) * P < 0.05;** P < 0.05; *** P < 0.001). Gel spots which showed significant differences in their volume between the control and lovastatin-treated cells were cut-out, proteins were digested and analyzed using liquid chromatography (LC) mass spectrometry (MS)/MS analysis. In MDAMB231 cells they were identified as GTPase-activating protein SH3-domain-binding protein 1 (G3BP1), TNF type 1 receptor-associated protein (TRAP1) and glutathione S-transferase (GST) omega proteins (Table 1a), whereas the spot belonging to citrate lyase beta and sterol carrier protein 2 (SCP-2) originated from MDAMB468 cells (Table 1b). Cofilin1/2 was identified as upregulated in both cell lines. The image and changes as observed in MDAMB231 cells is shown.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Expressing, Two-Dimensional Gel Electrophoresis, Standard Deviation, Control, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Western blot analysis of proteins involved in small GTPase-mediated cell signaling . Breast cancer cell lines MDAMB231 and MDAMB468 were treated with 8 μg/mL lovastatin lactone (Lova Lac) or lovastatin acid (Lova Ac) for 48 hours. For key proteins, western blot analysis was performed based on MDAMB231 cell extracts (for Ras homolog gene family member A (RhoA), cell division cycle 42 (CDC42) and GTPase-activating protein SH3-domain-binding protein 1 - phospho form (pG3BP1)), otherwise both cell lines are shown. Densitometry data were normalized based on the amount of β-actin. Data are presented as means ± standard deviations (n = 3) * P < 0.05;** P < 0.05; *** P < 0.001). Gel images were cropped to improve the clarity and conciseness of the presentation. GDI-2, Rho GDP dissociation inhibitor 2.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Western Blot, Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Western blot analysis of proteins involved in regulation of the cell cycle including the modulation of the E2F1-Rb activity . Breast cancer cell lines MDAMB231 and MDAMB468 were treated with 8 μg/mL lovastatin lactone (Lova Lac) or lovastatin acid (Lova Ac) for 48 hours. Western blot analysis of prohibitin was performed based on MDAMB231 cell extracts, otherwise both cell lines are shown. Densitometry data were normalized based on the amount of β-actin. Data are presented as means ± standard deviations (n = 3) * P < 0.05;** P < 0.05; *** P < 0.001. Gel images were cropped to improve the clarity and conciseness of the presentation. HMGB1, high-mobility group box 1; MCM7, minichromosome maintenance protein 7; MSH2, MutS homolog 2.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Western Blot, Activity Assay

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Western blot analysis of proteins involved in regulation of apoptosis and AKT-signaling . Breast cancer cell lines MDAMB231 and MDAMB468 were treated with 8 μg/mL lovastatin lactone (Lova Lac) or lovastatin acid (Lova Ac) for 48 hours. For key proteins, western blot analysis of phosphatase and tensin homolog (PTEN), pAkt and N-myc downstream regulated gene 1 (NDRG1) was performed based on MDAMB231 cell extracts, otherwise both cell lines are shown. Densitometry data were normalized based on the amount of β-actin. Data are presented as means ± standard deviations (n = 3) * P < 0.05;** P < 0.05; *** P < 0.001). Gel images were cropped to improve the clarity and conciseness of the presentation. PCNA, proliferating cell nuclear antigen.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Intracellular concentrations (nmol/g cell weight) of 13 C-labeled endogenous metabolites (glycolysis and TCA cycle intermediates, glucose) and lipid metabolites (choline-containing phospholipids, cholesterol)

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Changes in intracellular 13 C-labeled -alanine, -lactate, -glucose and -glutamine signals in MDAMB468 cells treated with 8 μg/mL lovastatin acid for 48 hours . 13 C-NMR spectra with embedded, corresponding 1 H-NMR spectra are shown (including citrate at 2.52 + 2.69 ppm). Arrows indicate the direction of signal changes (increase or decrease). Ala, alanine; Gln, glutamine; Glu, glutamine; GSH, total glutathione; Lac, lactate.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Labeling

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Representative 1 H-NMR spectra of MDAMB468 lipid extracts . Cells were treated with 8 μg/mL lovastatin acid for 48 hours. Arrows indicate the direction of signal changes (decrease). Chol, cholesterol (C18 and C19, CH 3 ); Δ (δ), double bond; F, fatty acid side chain; F α , F β , protons in the fatty acid chain; Fmix: -(CH 2 )n-, tCho, total choline-containing phospholipids.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

doi: 10.1186/bcr2485

Figure Lengend Snippet: Schematic diagram summarizing the effects of lovastatin lactone and acid on signaling pathways as found in the present study . The solid arrows mark the directional change of proteins (up- or down-regulation). Doted arrows mark hypothesized change in protein expression/activity. CDC42, cell division cycle 42; G3BP1, GTPase-activating protein SH3-domain-binding protein 1; GDI-2, Rho GDP dissociation inhibitor 2; LIMK, LIM domain kinase; MAPK, mitogen-activated protein kinase; MCM7, minichromosome maintenance protein 7; MSH2, MutS homolog 2; NDRG1, N-myc downstream regulated gene 1; p21, cyclin-dependent kinase inhibitor 1A; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog; Rb, retinoblastoma protein; RhoA, Ras homolog gene family member A.

Article Snippet: Lovastatin (in its lactone and hydroxy acid form) was purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Techniques: Protein-Protein interactions, Expressing, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Structure-guided investigations into HMG-CoA reductase as a herbicide target

doi: 10.1101/2022.04.18.488698

Figure Lengend Snippet: a , Representative images from post-emergence treatment of a model dicot, A. thaliana , and pre-emergence treatment of the monocot E. tef , with statins: rosuvastatin (Ro.), pravastatin (Pr.), simvastatin (Si.), mevastatin (Me.), lovastatin (Lo.), fluvastatin (Fl.), atorvastatin (At.) and pitavastatin (Pi.). b , A . thaliana (green) and E . t ef (grey) treated with a range of statins at 62.5 μM pre- and post-emergence on soil. Inhibition was quantified using green pixel area and plotted as a percentage of no-inhibitor control. n = 3 replicates with the mean ± standard deviation (s.d.).

Article Snippet: Treatments were conducted with rosuvastatin, simvastatin, fluvastatin, atorvastatin (AK Scientific), pravastatin (BOC Sciences), lovastatin (Sapphire Bioscience), mevastatin, pitavastatin (Focus Bioscience), RoundUp ® and atorvastatin analogues ( 1 - 9 ).

Techniques: Inhibition, Control, Standard Deviation

Journal: bioRxiv

Article Title: Structure-guided investigations into HMG-CoA reductase as a herbicide target

doi: 10.1101/2022.04.18.488698

Figure Lengend Snippet: Comparison of the sequences of AtHMG1 and A . terreus NIH2624 putative HMGR housekeeping gene (ATEG_02145) with A . terreus NIH2624 reported HMGR self-resistant gene – (ATEG_09965) from the lovastatin biosynthetic gene cluster reveals several residues potentially conferring HMGR with statin resistance HMGR (black stars). AtHMG1 active site-delineating residues shown with yellow boxes. Sequence numbers shown for AtHMG1 residue L558 is highlighted with two stars. Conserved regions (red box), highly similar residues (red text) .

Article Snippet: Treatments were conducted with rosuvastatin, simvastatin, fluvastatin, atorvastatin (AK Scientific), pravastatin (BOC Sciences), lovastatin (Sapphire Bioscience), mevastatin, pitavastatin (Focus Bioscience), RoundUp ® and atorvastatin analogues ( 1 - 9 ).

Techniques: Comparison, Sequencing, Residue

Journal: bioRxiv

Article Title: Structure-guided investigations into HMG-CoA reductase as a herbicide target

doi: 10.1101/2022.04.18.488698

Figure Lengend Snippet: a , The hydrophobic pocket in AtHMG1 delineated by labelled residues (blue sticks) with pitavastatin (magenta sticks) bound, illustrating L558 proximity to the hydrophobic ring of statins. b , AtHMG1 with the L558T mutation (grey bar) retained activity in vitro in the presence of statins: rosuvastatin (Ro.), pravastatin (Pr.), simvastatin (Si.), mevastatin (Me.), lovastatin (Lo.), fluvastatin (Fl.), atorvastatin (At.) and pitavastatin (Pi.), at 500 nM. n = 3 independent reactions with the mean ± s.d. c , In vitro inhibition of WT and L558T AtHMG1 by rosuvastatin revealed the L558T mutation conferred >20-fold resistance. n = 3 independent reactions with the mean ± s.d.

Article Snippet: Treatments were conducted with rosuvastatin, simvastatin, fluvastatin, atorvastatin (AK Scientific), pravastatin (BOC Sciences), lovastatin (Sapphire Bioscience), mevastatin, pitavastatin (Focus Bioscience), RoundUp ® and atorvastatin analogues ( 1 - 9 ).

Techniques: Mutagenesis, Activity Assay, In Vitro, Inhibition