Journal: Cell Death & Disease

Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

doi: 10.1038/s41419-018-0306-6

Figure Lengend Snippet: a Dose-dependent infection of hAECN with various A- and B-type rhinoviruses (HRV-A16, HRV-B14, HRV-B37, HRV-A2, HRV-A1A), and CVB3 and correlation with cell number. Values are means ± SD, n = 3. b Electron micrographs of hAECN infected with HRV-A16 (MOI 1) or treated with poly I:C or puromycin for 15 h, or uninfected cells. Proximity of vesicular structures with the cell surface indicated by black arrowheads, and swollen mitochondrial cristae in infected cells highlighted by arrows. c ROS production of HRV-A16 or HRV-A1A (MOI 1) infected cells 24 h pi, compared to luprox- (100 μ m , 1 h) or menadione (100 μ m , 1 h)-treated cells. d FACS analysis of annexin V stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. e Western blots against lamin A/C and beta-tubulin from lysates of hAECN cells infected with HRV-A16 (MOI 1, 15 h) or treated with puromycin (5 µg/ml, 15 h) with or without pan-caspase inhibitor QVD (5 µ m ). M r denotes relative molecular weight in kDa. f FACS analysis propidium iodide (PI) stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. g Western blot analysis of caspase 3, 7, 8, 9 activations in hAECN after infection with HRV-A16 (MOI 1, 15 h), poly I:C transfection (5 μg/ml) or external poly I:C addition (ext, 50 μg/ml). Arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. Infection is indicated by blotting against 3Cpro using beta-tubulin as a loading control.

Article Snippet: Lysates equivalent to 1.0×10 6 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

Techniques: Infection, Western Blot, Molecular Weight, Transfection

Journal: Cell Death & Disease

Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

doi: 10.1038/s41419-018-0306-6

Figure Lengend Snippet: a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).

Article Snippet: Lysates equivalent to 1.0×10 6 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

Techniques: Western Blot, Infection, Transfection, Expressing, Incubation, Staining, Fluorescence, Microscopy, Activation Assay

Journal: Cell Death & Disease

Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

doi: 10.1038/s41419-018-0306-6

Figure Lengend Snippet: a Linear representation of human RIPK1 comprising amino acids 1–671. The kinase domain is depicted in green, the middle domain (yellow) comprising an RHIM domain (gray) is responsible for p62/SQSTM1 binding, and the C-terminal death domain (DD, turquoise) links RIPK1 to cell death pathways. The predicted 3C consensus sequences are marked by purple lines. Antibody binding domains are highlighted by blue lines and numbers. b Time-resolved analyses of HeLa cells infected with HRV-A16 (MOI 1) by western blots against N-terminal RIPK1 (aa190), VP2, 3C, and GAPDH. Virus-specific RIPK1 processing products are highlighted by black arrows, and viral precursor proteins 3CD, 3ABC, and VP0 are indicated on the right side. c Virus-specific RIPK1 processing in primary hAECN independent of caspase. Western blots are shown against RIPK1 (leucine 190) and beta-tubulin from lysates of hAECN cells infected with HRV-A16 (MOI 1, 15 h) or treated with puromycin (5 µg/ml, 15 h) with or without pan-caspase inhibitor QVD (5 µ m ). d Comparison of RIPK1 processing in infected and poly I:C-treated cells. Western blots against N-terminal RIPK1 (leucine 190), middle domain RIPK1 (amino acids 165–402), C-terminal RIPK1 (amino acids 465–671), and beta-tubulin from lysates of HeLa cells after HRV-A16 infection (MOI 1, 15 h) or poly I:C transfection (5 µg/ml, 15 h). N-terminal virus-induced products are highlighted by black arrows. Poly I:C-induced RIPK1 products are indicated by white arrows. Star represents an unidentified product also present in uninfected cells. e A range of rhinoviruses including A16, A1A, B14, A2, and B37 induces cleavage of RIPK1 in hAECN and HeLa cells. Western blots of lysates from infected (MOI 1, 15 h) or uninfected hAECN (left panel) or HeLa cells (right panel) probed with N-terminal anti-RIPK1 antibody (L190) and anti-beta tubulin (b-Tub) antibody. Capital “M” denotes major HRV types tropic for ICAM-1, small “m” denotes minor types tropic for LDLR and related proteins.

Article Snippet: Lysates equivalent to 1.0×10 6 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

Techniques: Binding Assay, Infection, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

doi: 10.1038/s41419-018-0306-6

Figure Lengend Snippet: a Western blots against RIPK1 (leucine 190), VP2, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) and treated with pan-caspase inhibitor QVD (5 µ m , 15 h), calpain/cathepsin inhibitor E64d (10 µ m , 15 h), necrostatin-1 (Nec1, 1 µ m , 15 h), AG7088 (rupintrivir, 20 n m , 15 h) or drug-combinations. The 60 kDa RIPK1 fragment is highlighted by black arrow. b In vitro cleavage of RIPK1 by 3Cpro. HeLa cell extracts were incubated with recombinant 3C protease (r3C) with or without the 3C inhibitor AG7088 (20 n m ), and analyzed by western blotting against RIPK1 (leucine 190) and lamin A/C. Reference samples included lysates from HeLa infected with HRV-A16 (MOI 1, 15 h). RIPK1 cleavage is highlighted by black arrows. c GST-RIPK1 cleavage in vitro. Recombinant GST-RIPK1 was incubated with r3C in presence or absence of AG7088 (20 n m ) or QVD (5 µ m ), and reaction products analyzed by western blotting against N-terminal RIPK1 (leucine 190), middle domain RIPK1 (amino acids 133–422), and GST. d Late addition of AG7088 inhibits production of HRV-A16. HeLa cells were infected with HRV-A16 at three different MOI (log −1, −2, −3) and treated with AG7088 at different times pi. The titer of viral progeny was determined from cells and supernatants 24 h pi for each time point by end point titration assays. Data points represent mean values from three different experiments, including SD.

Article Snippet: Lysates equivalent to 1.0×10 6 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

Techniques: Western Blot, Infection, In Vitro, Incubation, Recombinant, Titration

Journal: Cell Death & Disease

Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

doi: 10.1038/s41419-018-0306-6

Figure Lengend Snippet: a Comparison of the RHIM domain from human RIPK1, RIPK3, 3Cpro from HRV-A16/B14 by sequence alignment. Yellow indicates amino acid identities, gray similarities, * denotes conserved amino acids in the RHIM domains of RIPK1/3. b Pro-caspase-8 immuno-precipitations, and western blots against Pro-caspase-8, RIPK1 (amino acids 190) and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), or poly I:C (5 µg/ml)-treated cells, and uninfected samples. Input lysates and immuno-precipitations are shown. LC denotes light chain. c RIPK1 immuno-precipitations (leucine 190) and western blot against 3Cpro from HRV-A16-infected HeLa cells (MOI 1, 15 h) or uninfected cells. HC denotes heavy chain. d RIPK1 immuno-precipitations (leucine 190) and western blots against RIPK1, cCasp8, and p62/SQSTM1 from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) or transfected with poly I:C (5 µg/ml). Input lysates and immuno-precipitations are shown. HC denotes heavy chain, IP immuno-precipitation. Arrow highlights HRV-A16-specific p62/SQSTM1 processing.

Article Snippet: Lysates equivalent to 1.0×10 6 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

Techniques: Sequencing, Western Blot, Infection, Transfection, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

doi: 10.1038/s41419-018-0306-6

Figure Lengend Snippet: a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.

Article Snippet: Lysates equivalent to 1.0×10 6 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

Techniques: Western Blot, Infection, Transfection, Expressing, Activation Assay, Binding Assay

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: IC 50 values of T. polycephalum leaves extracts on nine different cell lines after 48 h treatment.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques:

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: The tested agent induced cell cytotoxicity on MCF7 cells in a time-dependent manner. The IC 50 value of TPHE at 24, 48 and 72 h on the MCF7 cell line was determined to be 24.65 ± 2.41, 6.42 ± 0.35 and 5.16 ± 1.6 μg/mL, respectively. The data are shown as the mean ± SD ( n = 3).

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques:

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: Apoptotic morphological changes of MCF7 cells were observed by fluorescent microscope (×20 and ×40). ( A , E ) Untreated MCF7 cells demonstrated normal structure without prominent apoptosis after 72 h. ( B , F ) Early apoptotic features, including chromatin condensation and cell blebbing were observed after 24 h represented by intercalated AO dye (bright green). ( C , G ), ( D , H ) After 48 and 72 h, clear hallmarks of late apoptosis namely, blebbing and fragmented DNA were detected by orange color of PI. BL: Membrane blebbing; CC: Chromatin condensation; LA: Late apoptosis; VI: Viable MCF7 cells. * represents significant difference ( p < 0.05) compared with the control.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Microscopy, Membrane, Control

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: MCF7 cells were treated with IC 50 concentration of TPHE for 24 h. ( A ) Untreated cells were shown as control indicating viable cells. After treatment, the translocation of PS residues to the outer membrane of stained cells with Annexin-V-FITC shown in light green proved the induction of early apoptosis observed using fluorescence microscope with ( B ) ×20 and ( C ) ×40 magnifications.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Concentration Assay, Control, Translocation Assay, Membrane, Staining, Fluorescence, Microscopy

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: Effect of TPHE studied on cell cycle distribution of MCF7 cells. ( A ) After treatment with IC 50 concentration of TPHE for 24, 48 and 72 h, flow cytometry was used to analyze the cell cycle. ( B ) The result demonstrated the cell cycle arrest at G 1 phase. The percentage of apoptotic cells indicated the subG 1 population after 24, 48 and 72 h treatment. The data are shown as the mean ± SD ( n = 3). * represents significant difference ( p < 0.05) compared with the control.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Concentration Assay, Flow Cytometry, Control

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: The luminescence assay demonstrated the activities of caspase-7, -8 and -9 on MCF7 cells treated with TPHE for 3, 6, 12, 24 and 48 h. The data are shown as the mean ± SD ( n = 3). * represents significant difference ( p < 0.05) compared with the control.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Luminescence Assay, Control

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: Representative images of MCF7 cells (×20) treated with TPHE at IC 50 concentration. Cells stained with Hoechst 33342, cell membrane permeability, cytochrome c and MMP dyes. MCF7 cells produced a noteworthy reduction in MMP and a marked increase in chromatin condensation, cell membrane permeability and cytochrome c release.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Concentration Assay, Staining, Membrane, Permeability, Produced

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: The analysis of TPHE mediated apoptosis markers. Changes in cell membrane permeability, cytochrome c localization, MMP and total nuclear intensity were all quantified simultaneously in MCF7 cells. Treatment with TPHE caused a significant elevation in cell membrane permeability, nuclear intensity and cytochrome c release associated with loss of MMP. The data are shown as the mean ± SD ( n = 3). * represents significant difference ( p < 0.05) compared with the control.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Membrane, Permeability, Control

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: Effect of TPHE on mRNA expression of Bax and Bcl-2 in MCF7 cells. ( A ) Expression of Bcl-2 and Bax compared to β-actin in MCF7 cell line after treatment with TPHE shown in gel electrophoresis. ( B ) Time-dependent effect of THPE on Bax/Bcl-2 ratio in MCF-7 cells showed significant changes after 24, 48 and 72 h. The data are shown as the mean ± SD ( n = 3). * represents significant difference ( p < 0.05) compared with the control.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Expressing, Nucleic Acid Electrophoresis, Control

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: Immunofluorescent detection of Bcl-2 in MCF7 cells. The experiment was performed twice for this protein. Data revealed a decrease in the level of Bcl-2 protein after treatment. ( A ) Composite image of FITC with DAPI, ( B ) FITC conjugated with Bcl-2 and ( C ) DAPI.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques:

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: Immunofluorescent detection of Bax in MCF7 cells. The experiment was performed twice for this protein. Data revealed an increase in the level of Bax protein after treatment. ( A ) Composite image of FITC with DAPI, ( B ) FITC conjugated with Bax and ( C ) DAPI.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques:

Journal: Molecules

Article Title: Tanacetum polycephalum (L.) Schultz-Bip. Induces Mitochondrial-Mediated Apoptosis and Inhibits Migration and Invasion in MCF7 Cells

doi: 10.3390/molecules19079478

Figure Lengend Snippet: TPHE blocked MCF7 cell migration and invasion. ( A ) TPHE suppressed breast cancer cell migration. MCF7 cells were seeded in 6-well plates. The confluence MCF7 cells were wounded and imaged (0 h). After treatment of MCF7 cells with TPHE at IC 50 concentration, cells were incubated at 37 °C for 24, 48 and 72 h and photographed. ( B ) The untreated cells of MCF7 were significantly migrated within 72 h. ( C ) The quantitative analysis of images revealed the significant suppressive effect of TPHE against MCF7 migration. ( D ) TPHE significantly suppressed MCF7 cell invasion. Percentage of invasion was determined as the percent of MCF7 cells invaded through Matrigel inserts vs . the total MCF7 cells invaded through the control inserts. The data are shown as the mean ± SD ( n = 3). * represents significant difference ( p < 0.05) compared with the control.

Article Snippet: The MCF7 cells were incubated at 4 °C for 24 h. Then, Bcl-2 and Bax fluorochrome-conjugated secondary antibody diluted (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in antibody dilution were supplemented to the MCF7 cells and incubated for 1 h. After washing three times in PBS, cells were treated with DAPI prior to being examined using CellReporterTM Molecular Devices.

Techniques: Migration, Concentration Assay, Incubation, Control

Journal: Nature Communications

Article Title: A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host

doi: 10.1038/s41467-019-08869-9

Figure Lengend Snippet: Analysis of exotoxin A from Pseudomonas aeruginosa (EPA) glycosylation with the CPS8 capsular polysaccharide. Western blot analysis of EPA-CPS8 bioconjugates compared against EPA alone. a (Left panel Anti-His channel probing for Hexa-histidine-tagged EPA purified from SDB1 expressing CPS8 in the presence or absence of PglS. a (Middle panel) Anti-glycan channel probing for CPS8. a (Right panel) Merged images for left and middle panels. b EPA-CPS8 separated on a SDS- polyacrylamide gel stained with Coomassie. c , d Intact protein mass spectrometry analysis showing the MS1 mass spectra for purified EPA-CPS8. The EPA fusion protein has a theoretical mass of 79,526.15Da and can be observed as the peak at 79,514.76 Da. The EPA fusion protein was also observed in multiple states of increasing mass corresponding to the CPS8 repeating subunit, which has a theoretical mass of 662Da. Varying glycoforms of the EPA-CPS8 were observed and are denoted by “g numeric ”, where “g” stands for glycoform and the “numeric” corresponds to the number of repeating CPS8 subunits. The EPA fusion protein was modified with up to 11 repeating subunits of the CPS8 glycan. Panel d provides a zoomed in view of the varying EPA-CPS8 glycoforms. Source data are provided as a Source Data file

Article Snippet: Cell lysates containing the equivalent of OD 600 = 0.1 units were loaded on 12.5% or 7% in-house prepared sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, which were then transferred to nitrocellulose membranes (Bio-Rad).

Techniques: Western Blot, Purification, Expressing, Staining, Mass Spectrometry, Modification