Journal: Scientific Reports

Article Title: Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore

doi: 10.1038/s41598-019-41626-y

Figure Lengend Snippet: DDAO arachidonate structure and activation. DDAO arachidonate, consisting of DDAO esterified to arachidonic acid is non-fluorescent at 600 nm. Cleavage of the ester linkage by cPLA2α, releases free DDAO, which becomes fluorescent with an ex/em maxima of 600/660 nm.

Article Snippet: 4175-Luc+ cells were generously provided by Dr. Andy Minn . Human cPLA2α monoclonal rabbit antibody (Cat. # 2832S) and human beta-tubulin rabbit monoclonal antibody (Cat. # 2128S) were purchased through Cell Signaling Technology.

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore

doi: 10.1038/s41598-019-41626-y

Figure Lengend Snippet: DDAO arachidonate activation in cell lines varying in cPLA2α expression. ( A ) cPLA2α protein expression in four breast cancer cell lines representing three different molecular sub-types: Basal-like triple negative (4175-Luc+ and MDA-MB-231), HER2-overexpressing (SKBR3), and luminal-like (MCF-7). Membrane was cut and lower portion was blotted for ß-tubulin as a loading control. Full gel is provided in Fig. . ( B ) Fluorescence (ex/em: 685/720 nm) in cells treated with DDAO arachidonate or DDAO palmitate. cPLA2α inhibition through AVX235 treatment resulted in significant fluorescence reduction in 4175-Luc+ and MDA-MB-231 cells (p = 0.006 and 0.03, respectively). ( C ) Adjusted cPLA2α activity derived from ( B ) through the subtraction of DDAO palmitate fluorescence from DDAO arachidonate fluorescence, plotted relative to 4175-Luc+ activity. ( D ) Correlation of cPLA2α expression ( A ) to cPLA2α activity ( C ) showed a strong positive correlation (R 2 = 0.9633) between cPLA2α expression and activity.

Article Snippet: 4175-Luc+ cells were generously provided by Dr. Andy Minn . Human cPLA2α monoclonal rabbit antibody (Cat. # 2832S) and human beta-tubulin rabbit monoclonal antibody (Cat. # 2128S) were purchased through Cell Signaling Technology.

Techniques: Activation Assay, Expressing, Membrane, Control, Fluorescence, Inhibition, Activity Assay, Derivative Assay

Journal: Scientific Reports

Article Title: Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore

doi: 10.1038/s41598-019-41626-y

Figure Lengend Snippet: Confocal fluorescence microscopy of DDAO arachidonate and DDAO palmitate activation. ( A ) MDA-MB-231, SKBR3, and MCF7 cells treated with DDAO arachidonate or DDAO palmitate for four hours with and without cPLA2α inhibition. cPLA2α activated DDAO fluorescence is shown as a heat map with brighter areas representing higher fluorescence intensity. The highest fluorescence was observed in MDA-MB-231 cells, followed by SKBR3, then MCF-7. In all cases, cPLA2α inhibition appeared to decrease fluorescence. For all cell lines, DDAO palmitate resulted in considerably less fluorescence signal. ( B ) Subcellular distribution of DDAO arachidonate following four hours of probe incubation. DDAO fluorescence is shown in green, and nuclear stain (HOECHST 33342) is shown in blue. Fluorescence is not adjusted for relative intensity, but is optimized for visualization of probe distribution.

Article Snippet: 4175-Luc+ cells were generously provided by Dr. Andy Minn . Human cPLA2α monoclonal rabbit antibody (Cat. # 2832S) and human beta-tubulin rabbit monoclonal antibody (Cat. # 2128S) were purchased through Cell Signaling Technology.

Techniques: Fluorescence, Microscopy, Activation Assay, Inhibition, Incubation, Staining

Journal: Scientific Reports

Article Title: Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore

doi: 10.1038/s41598-019-41626-y

Figure Lengend Snippet: DDAO arachidonate fluorescence in cPLA2α knockdown 4175-Luc+ cells. ( A ) Protein expression in 4175-Luc+ cells, cPLA2α CRISPR knockout plasmid transfected cells, and scrambled CRISPR knockout plasmid transfected cells. Full gel is presented. ( B ) Quantitated cPLA2α protein levels, taken from ( A ), relative to beta-tubulin. Significant knockdown of cPLA2α protein was observed in cPLA2α CRISPR plasmid transfected cells (p = 8.2 × 10 −7 ). ( C ) Relative fluorescence of DDAO arachidonate or DDAO palmitate with time in cPLA2α knockdown cell lines. ( D ) Quantitated relative fluorescence at 120 minutes, taken from ( C ). Significant reduction of fluorescence was observed in cPLA2α knockdown cells treated with DDAO arachidonate (p = 1.58 × 10 −6 ). No significant changes in fluorescence were observed when treated with DDAO palmitate.

Article Snippet: 4175-Luc+ cells were generously provided by Dr. Andy Minn . Human cPLA2α monoclonal rabbit antibody (Cat. # 2832S) and human beta-tubulin rabbit monoclonal antibody (Cat. # 2128S) were purchased through Cell Signaling Technology.

Techniques: Fluorescence, Knockdown, Expressing, CRISPR, Knock-Out, Plasmid Preparation, Transfection

Journal: Journal of Neuroinflammation

Article Title: Early upregulation of cytosolic phospholipase A 2 α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1186/s12974-021-02326-5

Figure Lengend Snippet: Misfolded SOD1 accumulation precedes cPLA 2 α upregulation and activation. A A representative double immunofluorescence staining of cPLA 2 α (red) and misfolded SOD1 (B8H10, green) proteins in the lumbar spinal cord sections of WT and mutant SOD1 G93A mice during the development of the disease (3, 6 and 17 weeks). Scale bars = 500 μm (upper panel), 100 μm (lower panel) and inset 20 μm. The means ± SEM fluorescence intensity for both magnifications are presented in the bar graph as arbitrary units. Four mice for each time point and five fields for each mouse were analyzed. *** p < 0.001 compared to control mice (WT). n.s. non-significant. B A representative immunoblot analysis of cPLA 2 α, mutant SOD1 G93A and their corresponding calreticulin protein expression in the spinal cord lysates of WT and mutant SOD1 G93A mice during the development of the disease. cPLA 2 α protein expression was determined by dividing the intensity of each cPLA 2 α or mutant SOD1 G93A (SOD1 G93A ) by the intensity of the corresponding calreticulin band after quantitation by densitometry and expressed in the bar graph as arbitrary units. The bar graphs are the means ± SE of 4 mice in each group. *** p < 0.001 significance—compared to control mice (WT). n.s. non-significant. C . Misfolded SOD1 was immunoprecipitated using anti-B8H10 antibodies from spinal cord lysates of WT and mutant SOD1 G93A mice during the development of the disease. A representative immunoblot analysis of the misfolded SOD1 (IP B8H10, left) and 10% of the unbound (Unbound, right) fractions are shown. nc- negative control, immunoprecipitation without anti-B8H10 antibodies. D A representative immunofluorescence staining of phosphor-cPLA 2 α (p-cPLA 2 α) in the lumbar spinal cord sections of WT and mutant SOD1 G93A mice during the development of the disease (3, 6 and 17 weeks). Scale bars = 500 μm (upper panel) and 100 μm (lower panel). The means ± SEM fluorescence intensity for both magnifications are presented in the bar graph in arbitrary units. Four mice for each time point and five fields for each mouse were analyzed. *** p < 0.001 significance compared to WT mice. n.s. non-significant

Article Snippet: The detection of immunoreactive bands was carried out as described above but for cPLA 2 α, using 1:500 rabbit anti cPLA 2 α (GeneTex Inc, Alton Pkwy Irvine, CA, USA).

Techniques: Activation Assay, Double Immunofluorescence Staining, Mutagenesis, Fluorescence, Western Blot, Expressing, Quantitation Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Early upregulation of cytosolic phospholipase A 2 α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1186/s12974-021-02326-5

Figure Lengend Snippet: cPLA 2 α and misfolded SOD1 accumulates specifically in motor neurons of young pre-symptomatic SOD1 G93A mice. A Double staining (red) of motor neurons (ChAT), microglia (Iba-1) or astrocytes (GFAP) and misfolded SOD1 (B8H10, green) in the spinal cord sections of 6 weeks old pre-symptomatic SOD1 G93A mice. Scale bars = 20 μm. Shown a representative out of five mice. B Double staining (red) of motor neurons (ChAT), microglia (Iba-1) or astrocytes (GFAP) and cPLA 2 α (green) in the spinal cord sections of 6 weeks old pre-symptomatic SOD1 G93A mice. Scale bars = 20 μm. Shown a representative out of five mice. C Double immunofluorescence staining of cPLA 2 α (red) with misfolded SOD1 (B8H10, green) proteins in the spinal cord sections of control mice (WT) and 6 weeks old pre-symptomatic mutant SOD1 G93A mice. Scale bars = 100 μm. D The Pearson coefficient correlation (r) between cPLA 2 α and misfolded SOD1 in the motor neurons of the intensity of cPLA 2 α and misfolded SOD1 expressed in arbitrary units of immunostaining as presented in the representative results in C. Four filed in each of the 8 different mice analyzed

Article Snippet: The detection of immunoreactive bands was carried out as described above but for cPLA 2 α, using 1:500 rabbit anti cPLA 2 α (GeneTex Inc, Alton Pkwy Irvine, CA, USA).

Techniques: Double Staining, Double Immunofluorescence Staining, Mutagenesis, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Early upregulation of cytosolic phospholipase A 2 α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1186/s12974-021-02326-5

Figure Lengend Snippet: Accumulation of misfolded SOD1 in primary motor neurons induced cPLA 2 α upregulation. A Immunoblot analysis of human SOD1 WT or mutant SOD1 G93A (hSOD1), expressed in primary motor neurons by infection of AAV1/2 as described in materials and methods. Immunoblot analysis of mouse SOD1 (mSOD1) is shown as a control. B Light microscope pictures of primary motor neurons. Control—without infection, motor neurons expressing human wild type SOD1 (SOD1 WT) or mutant SOD1 (SOD1 G93A). Scale bars = 50 μm. C Double immunofluorescence staining of cPLA 2 α (green) with misfolded SOD1 (B8H10, red) in motor neurons expressing SOD1 WT , mutant SOD1 G93A and control cells. Two upper panels scale bars = 50 μm and two lower panels scale bars = 20 μm. 3 different independent experiments were analyzed and showed similar results. The means ± SEM fluorescence intensity for cPLA 2 α and misfolded SOD1 is presented in the bar graphs as arbitrary units. Five fields in each of the 3 different treatments of motor neurons in each experiment was analyzed. Significance compared to control *** p < 0.001, n.s. non-significant

Article Snippet: The detection of immunoreactive bands was carried out as described above but for cPLA 2 α, using 1:500 rabbit anti cPLA 2 α (GeneTex Inc, Alton Pkwy Irvine, CA, USA).

Techniques: Western Blot, Mutagenesis, Infection, Light Microscopy, Expressing, Double Immunofluorescence Staining, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Early upregulation of cytosolic phospholipase A 2 α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1186/s12974-021-02326-5

Figure Lengend Snippet: Partial binding between cPLA 2 α and mutant SOD1 G93A in motor neurons of 6 weeks old mutant SOD1 G93A mice. A Immunoprecipitation with antibody against cPLA 2 α and Western Blot analysis for cPLA 2 α and mutant SOD1 in the spinal cord lysate of control (WT) and mutant SOD1 G93A mice during the development of the disease shown by a representative immunoblot. Negative control (n.c). -without antibodies against cPLA 2 α. B Motor performance on accelerating Rotarod test ( n = 10 mice). The arrows show the immunoprecipitation analysis along the development of the disease. C Airyscan detector high resolution confocal microscopy images of double immunofluorescence staining of cPLA 2 α (red) with misfolded SOD1 (B8H10, green) proteins in the spinal cord sections of 6 weeks old pre-symptomatic SOD1 G93A mouse. Scale bars = 20 μm. D The coupling of both proteins (cPLA 2 α and misfolded SOD1) in the line shown by the arrow was analyzed and presented as intensity along the line. The ellipse shows an example of binding between both proteins

Article Snippet: The detection of immunoreactive bands was carried out as described above but for cPLA 2 α, using 1:500 rabbit anti cPLA 2 α (GeneTex Inc, Alton Pkwy Irvine, CA, USA).

Techniques: Binding Assay, Mutagenesis, Immunoprecipitation, Western Blot, Negative Control, Confocal Microscopy, Double Immunofluorescence Staining

Journal: Journal of Neuroinflammation

Article Title: Early upregulation of cytosolic phospholipase A 2 α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1186/s12974-021-02326-5

Figure Lengend Snippet: Elevated cPLA 2 α protein expression in the motor neurons is highly correlated with TNFα. A Double immunofluorescence staining of cPLA 2 α (green) and TNFα (red) proteins in the lumbar spinal cord sections of WT and 6 weeks old mutant SOD1 G93A mice. Scale bars = 100 μm. B The Pearson coefficient correlation (r) between cPLA 2 α and TNFα in the spinal motor neurons of mutant SOD1 G93A mice was analyzed. Florescence intensity is expressed in arbitrary units of immunostaining as presented in the representative results in A. Four fields in each of the 8 different mice analyzed. C Elevated cPLA 2 α and TNFα in primary motor neurons expressing mutant SOD1 G93A . Double immunofluorescence staining of cPLA 2 α (green) and TNFα (red) in primary motor neurons expressing human SOD1 WT , mutant SOD1 G93A and control cells described in Fig. . Two upper panels, scale bars = 50 μm and two lower panels, scale bar s = 20 μm. 3 different independent experiments were analyzed and showed similar results. The means ± SEM fluorescence intensity for cPLA 2 α and TNFα is presented in the bar graphs as arbitrary units. Five fields in each of the 3 different treatments of motor neurons in each experiment was analyzed. Significance compared to control *** p < 0.001, n.s. non-significant

Article Snippet: The detection of immunoreactive bands was carried out as described above but for cPLA 2 α, using 1:500 rabbit anti cPLA 2 α (GeneTex Inc, Alton Pkwy Irvine, CA, USA).

Techniques: Expressing, Double Immunofluorescence Staining, Mutagenesis, Immunostaining, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Early upregulation of cytosolic phospholipase A 2 α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

doi: 10.1186/s12974-021-02326-5

Figure Lengend Snippet: Secreted TNFα is responsible for cPLA 2 α upregulation. A Representative light microscope pictures of undifferentiated and differentiated motor like NSC34 cells, scale bars = 50 μm. B cPLA 2 α upregulation induced by mutant SOD1 G93A expressing cells is prevented in the presence of anti-TNFα antibodies. NSC-34 cells were transfected with human SOD1 WT or mutant SOD1 G93A plasmids for 72 h before lysates preparations. TNFα neutralizing antibodies were added at 48 h. A representative immunoblot analysis of cPLA 2 α in NSC34 cells transfected with human SOD1 WT (WT) or mutant SOD1 G93A (G93A) in the absence or presence of anti-TNFα. Control cells without transfection (Cont.). cPLA 2 α protein expression was determined by dividing the intensity of each cPLA 2 α with the intensity of the corresponding human SOD1 or mouse SOD1 after quantitation by densitometry and expressed in the bar graph as arbitrary units. The bar graphs are the means ± SE of three experiments. Significance compared to control *** p < 0.001, n.s. non-significant. C Representative confocal pictures of immunofluorescence staining of TNFRI and TNFRII in undifferentiated and differentiated motor like NSC34 cells, scale bars = 20 μm. Two other experiments showed similar results. D A representative immunoblot analysis of a dose dependent effect of TNFα (0.005–0.1 ng/ml) for 24 h on cPLA 2 α expression in differentiated motor neuron-like NSC34 cells. cPLA 2 α protein expression was determined by dividing the intensity of each cPLA 2 α with the intensity of the corresponding actin after quantitation by densitometry and expressed in the bar graph as arbitrary units. The bar graphs are the means ± SE of 3 experiments. Significance compared to control *** p < 0.001, n.s. non-significant. E A representative immunofluorescence staining of a dose dependent effect of TNFα (0.05–0.1 ng/ml) for 24 h on cPLA 2 α expression in differentiated motor neuron-like NSC34 cells. Scale bars = 50 μm. The bar graph is the mean ± SE of 3 different experiments. Significance compared to control *** p < 0.001, n.s. non-significant

Article Snippet: The detection of immunoreactive bands was carried out as described above but for cPLA 2 α, using 1:500 rabbit anti cPLA 2 α (GeneTex Inc, Alton Pkwy Irvine, CA, USA).

Techniques: Light Microscopy, Mutagenesis, Expressing, Transfection, Western Blot, Quantitation Assay, Immunofluorescence, Staining

Journal: Biomolecules & Therapeutics

Article Title: Curcumin Inhibits the Activation of Immunoglobulin E-Mediated Mast Cells and Passive Systemic Anaphylaxis in Mice by Reducing Serum Eicosanoid and Histamine Levels

doi: 10.4062/biomolther.2013.092

Figure Lengend Snippet: Effect of curcumin on the Syk pathway. IgE-sensitized BMMCs were preincubated with curcumin or Bay 61-3606 for 1 h, and then stimulated with DNP-HSA for 5 min. Cell lysates were subjected to immunoprecipitation and immunoblot analysis for the phosphorylated forms of Syk, LAT and PLCγ1. Bay 61-3606 was used as a positive control with respect to the suppression of the Syk-mediated pathway. The relative ratios of p-Syk/Syk, p-LAT/LAT and p-PLCγ1/PLCγ1 protein levels were determined by measuring immunoblot band intensities by scanning densitometry ( ** p <0.01 and *** p <0.001). The results shown are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibodies for phospho-cPLA 2 α (Ser505), cPLA 2 α, 5-LO, PLCγ1, IKKα/β lamin B and NF-κB p65 as well as secondary goat anti-rabbit IgG-HRP and rabbit anti-goat IgG-HRP antibodies, total Syk, total LAT, and Bay 61-3606 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and antibodies for phosphotyrosine was purchased from Millipore (Millipore, Billerica, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Positive Control

Journal: Biomolecules & Therapeutics

Article Title: Curcumin Inhibits the Activation of Immunoglobulin E-Mediated Mast Cells and Passive Systemic Anaphylaxis in Mice by Reducing Serum Eicosanoid and Histamine Levels

doi: 10.4062/biomolther.2013.092

Figure Lengend Snippet: Effect of curcumin on the Syk pathway. IgE-sensitized BMMCs were preincubated with curcumin or Bay 61-3606 for 1 h, and then stimulated with DNP-HSA for 5 min. Cell lysates were subjected to immunoprecipitation and immunoblot analysis for the phosphorylated forms of Syk, LAT and PLCγ1. Bay 61-3606 was used as a positive control with respect to the suppression of the Syk-mediated pathway. The relative ratios of p-Syk/Syk, p-LAT/LAT and p-PLCγ1/PLCγ1 protein levels were determined by measuring immunoblot band intensities by scanning densitometry ( ** p <0.01 and *** p <0.001). The results shown are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibodies for phospho-cPLA 2 α (Ser505), cPLA 2 α, 5-LO, PLCγ1, IKKα/β lamin B and NF-κB p65 as well as secondary goat anti-rabbit IgG-HRP and rabbit anti-goat IgG-HRP antibodies, total Syk, total LAT, and Bay 61-3606 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and antibodies for phosphotyrosine was purchased from Millipore (Millipore, Billerica, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Positive Control