Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 3. Induction of apoptosis in HL-60 cells treated with DL-247 for 24 h. (A) Representative scattered blots of flow cytometry analysis of apoptosis by double-staining with Annexin-V and PI. The percentage of viable cells is shown in quadrant Q3. Quadrant Q4 indicates the percentage of early apoptotic cells (Annexin V-positive cells), whereas quadrant Q2 shows the percentage of late apoptotic/death cells (Annexin V-and PI-positive cells). (B) Quantitative analysis of apoptotic cell death by Annexin V and PI assay. (C) Representative histograms of flow cytometry analysis of DNA fragmentation measured by TUNEL assay. (D) Quantitative analysis of DNA fragmentation. (B,D): Data are presented as mean ± SEM of three independent experiments. Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001 vs. control.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Cytometry, Double Staining, TUNEL Assay, Comparison, Control

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 5. Analysis of typical features of intrinsic apoptosis pathway in HL-60 cells treated with DL-247 for 24 h. (A) Percentage of phosphorylated H2AX positive cells measured by flow cytometry (Alexa Fluor 647 Mouse Anti-H2AX (pS139) antibody staining). (B) Mitochondrial membrane potential (∆Ψm) changes measured by flow cytometry (JC-1 staining). FCCP (30 µM, 30 min incubation at 37 ◦C) was used as a positive control. (C) Bax and Bcl-2 gene expression changes analyzed by real-time PCR. (D) Intracellular ROS generation measured by flow cytometry (CellROX Green Reagent staining). Menadione (200 µM, 1 h incubation at 37 ◦C) was used as a positive control. (A–D): Data are presented as mean ± SEM of three independent experiments. (A,B,D): Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. * p < 0.05, *** p < 0.001 vs. control. C: Statistical significance was assessed using Student-t test. * p < 0.05, *** p < 0.001 vs. control.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Cytometry, Staining, Membrane, Incubation, Positive Control, Gene Expression, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 6. Changes in Fas concentration in HL-60 cells treated with DL-247 for 24 h analyzed by Human Fas ELISA kit. Data are presented as mean ± SEM of three independent experiments. Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001 vs. control.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 9. Multiparameter analysis (proliferation, DNA damage and apoptosis) of the synergistic effect of Tx (9 nM) in combination with DL-247 (1.15 µM) in HL-60 cells. Panel (A): DAPI vs. BrdU PerCP-Cy™5.5 staining profile. BrdU positive cells are in the inside frame. Panel (B): cleaved PARP (Asp214) PE vs. H2AX (pS139) Alexa profile. Squares Q1 + Q2 represent H2AX (pS139) Alexa positive cells, squares Q2 + Q4 represent Cleaved PARP (Asp214) PE positive cells, whereas square Q3 represents H2AX (pS139) Alexa and Cleaved PARP (Asp214) PE negative cells.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Staining

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: Protective genes.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Membrane

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: (A) Forward and side scatter plot of lung cells. Circles show the lymphocyte, macrophages and neutrophil populations. To the right a single color histograms showing expression of receptor CD46 from representative control, emphysema and end-stage participants. Pooled data from all participants (control, n = 4; emphysema, n = 4, end-stage n = 5) showing percent (median±SD) of total lung neutrophils, and macrophages expressing CD46; Cumulative values for lymphocytes showed a significative decrease (control, n = 6; emphysema, n = 7, end-stage n = 6, p<0.05) more patient were included with the same lung characteristics. (B) Gene expression of Casp 8, on the same patients, determined by qRT-PCR (median±SD) on emphysema patients with (n = 5) and without cancer (n = 4) shows no different expression level due to cancer away from the emphysemic region (p = 1). Mann-Whitney test was used to determine significant difference.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Expressing, Control, Gene Expression, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: (A) Increased expression of CD46 in the lung tissue of no smoker mice compared to smoke exposed mice determined by IHC, arrow heads, and by western western blot on lung tissue homogenates of control mice or smoke-exposed mice (p = 0.02, n = 4, n = 4). (B) Increased deposition of C3b on lung tissue of control mice (n = 3) compared to smoke- exposed mice at 4 and 24 weeks (n = 3) determined by Western blot. Middle plot, immunoprecipitation of C3b from lung homogenate shows a significant increase of C3b deposition upon smoke exposure (p = 0.05). Elastin was stained and detected after stripping the membrane, showing significant increased co-precipitation with C3b (p = 0.01). Student t-test two tails was used to compare both groups; values in the plots represent average ± SD.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Staining, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: (A) CD46 coupling with CD3 activates the transcription factor ubiquitously transcribed tetratricopeptide (UTX), which interacts with signal transducer and activator of transcription 1 (STAT1) both translocate to the nucleus to activate proliferation of CD4 + T cells. T cell receptor activates Bcl-2 that inhibits cytochrome C (Cyto C) released by chloride intracellular channel 4 (CLIC4), which positively regulates Fas Ligand receptor (CD95) mediated activation of Caspase 8 (Casp 8) and apoptosis. Homeodomain interacting protein kinase 3 (HIPK3) phophorilates CD95, and HuR stabilizes CD95 mRNA also positively regulates it. While 2 negative regulators of CD95 are methionine adenosyl transferase II, alpha (MATII) and hepatocyte growth factor receptor (HFGR). Red font signifies protective gene, black arrow apoptosis activation, black bold arrow apoptosis inhibition. Red arrow, proliferation pathway. Red proteins are kinases; blue, transcription factors; light brown, cell surface receptors. (B) Total CD46 expression on the surface of lung cells of patients (n = 14) significantly correlates with their FEV1% in a linear regression using minimum square approximation, with a coefficient r = 0.563 and a goodness of fit p = 0.036. The FasL receptor, CD95, protein expression on the same patients, also, positively correlates with FEV1% with an r = 0.711 and a goodness of fit p = 0.006. There is a significative linear association between cells surface proteins expression, CD46 + and CD95 + (r = 0.666, p = 0.012). Right top plot, total lung lymphocytes CD4 + plus CD8 + has a constant number in the lung parenchyma despite their lung FEV1% change with disease progression (r = 0.107, p = 0.6332, n = 22) although there is a significant change in the number of CD4 + and CD8 + T cells. Right middle plot, in the same patients group, CD4 + T cells (black circles) correlates directly with the FEV1% (r = 0.549, slope = 0.24, p = 0.008, n = 22), conversely CD8 + T cells (red circles) inversely correlates with FEV1% increasing its number with disease progression (r = 0.480, slope = −0.329, p = 0.024). Bottom right plot shows a direct correlation between FEV1% and depletion of CD4 + with increment of CD8 + T cells (slope = 8.4, r = 0.726, p = 0.0002). (C) There is a remarkable association among CD46 + decrement and CD4 + /CD8 + ratio of T cells, r = 0.896 p = 0.006, n = 7. (D) Increased IgG to elastin in early-onset COPD (EO-COPD) patients, relative to normal controls, tested in n = 21 and n = 28, respectively, plot represents the patients that give signal above the threshold n = 9 for both EO-COPD and control with a significant increased in the diseased group p = 0.038, determined by Mann-Whitney test, values represent median ± SD.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Activation Assay, Inhibition, Expressing, Biomarker Discovery, Control, MANN-WHITNEY