Journal: The Journal of Biological Chemistry

Article Title: Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori , Dually Couples to the G q and G s Signaling Cascades *

doi: 10.1074/jbc.M112.441675

Figure Lengend Snippet: Corazonin-mediated cAMP formation in BNGR-A21-expressing cells. A , cAMP accumulation in HEK293 cells transiently co-transfected CRE-Luc ( CRE-L ) and BNGR-A21 with or without vehicle vector ( V ) was determined in response to corazonin treatment (1 μ m ). B , effects of PTX or CTX on corazonin-mediated stimulation of cAMP accumulation. HEK293 cells expressing BNGR-A21 were pre-treated with PTX (100 ng/ml) or CTX (300 ng/ml) overnight prior to incubation with coraoznin (1 μ m ) for 4 h. C , cAMP accumulation in Sf21 cells transiently co-transfected with CRE-Luc and FLAG-A21 was determined in response to DMEM (control) and corazonin treatments (1 μ m ). Effects of tachykinin-1 and neuropeptide F-1 on BNGR-A21-mediated stimulation of cAMP accumulation in HEK293 ( D ) and Sf21 ( E ) cells. cAMP accumulation was assayed in response to different doses of corazonin in HEK293 ( F ) and Sf21 ( G ) cells transiently co-transfected FLAG-A21/CRE-Luc. All data are shown as mean ± S.E. from at least three independent experiments. Data are expressed as the mean ± S.E. Data were analyzed by using the Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: H89, Go6983, U73122, U0126, cholera toxin (CTX), and pertussis toxin (PTX) were purchased from Tocris Bioscience.

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Control

Journal: The Journal of Biological Chemistry

Article Title: Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori , Dually Couples to the G q and G s Signaling Cascades *

doi: 10.1074/jbc.M112.441675

Figure Lengend Snippet: Effects of inhibitors PKA, PKC, and MEK on corazonin-mediated ERK1/2 activation in BNGR-A21-expressing cells. Concentration dependence ( A ) and time course ( B ) of corazonin-stimulated phosphorylation of ERK1/2 in stable BNGR-A21-expressing HEK293 cells. Concentration dependence ( C ) and time course ( D ) of corazonin-stimulated phosphorylation of ERK1/2 in Sf21 cells transfected with BNGR-A21. E , dose-dependent effects of pertussis toxin ( PTX , 100 ng/ml) and MEK inhibitor U0126 (1 μ m ) on BNGR-A21 mediated activation of ERK1/2. Dose-dependent ( F ) and time course ( G ) of PKA inhibitor H89 (10 μ m ) and PKC inhibitor Go6983 (10 μ m ) on BNGR-A21-mediated activation of ERK1/2. The cells were pretreated with or without (control) inhibitors for 1 h and then stimulated with corazonin (1 n m , 5 min). The p-ERK was normalized to a loading control ( total-ERK ). The data shown are representative of at least three independent experiments. Statistical analysis was performed by a two-tailed Student's t test (**, p < 0.01; ***, p < 0.001, versus counterpart control).

Article Snippet: H89, Go6983, U73122, U0126, cholera toxin (CTX), and pertussis toxin (PTX) were purchased from Tocris Bioscience.

Techniques: Activation Assay, Expressing, Concentration Assay, Phospho-proteomics, Transfection, Control, Two Tailed Test

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: HSF1A leads to a reduction in the levels of ADP-ribosylated Gαi within cells, while it has no impact on the enzymatic activity of PTS1. ( a ) CHO cells were subjected to pre-incubation with either HSF1A or VER for 1 h at 37 °C. In comparison, untreated cells (con) serve as the control. For further control, cells were treated with the solvent of both inhibitors, DMSO, at concentrations corresponding to the 200 µM HSF1A. Following this, the cells were exposed to 10 ng/mL PT for 4 h, both with and without the respective inhibitors. The cellular lysates were then examined to determine the ADP-ribosylation status of Gαi through incubation with 100 nM PTS1 in the presence of biotin-NAD + . The presence of biotin-labeled (ADP-ribosylated) Gαi was detected, and the results were normalized to the loading control, confirmed by Hsp90 detection. The values displayed indicate the signal percentage relative to untreated cells, and are normalized to the respective loading control, with mean ± SEM values obtained from at least five values across five independent experiments. Significance is evaluated using mixed-effects analysis and Dunnett’s multiple comparisons test, with values compared to samples treated solely with PT (white bar). ( b ) A549 cells were pre-incubated with HSF1A, or DMSO (corresponding to 200 µM HSF1A) as the control, for 30 min at 37 °C. Similar to panel ( a ), 10 ng/mL PT was introduced to the cells for a 4 h period in the presence, or absence, of the inhibitors. The lysates were then analyzed to assess the ADP-ribosylation status of Gαi. Comparable protein loading was again confirmed by Hsp90 detection, and the results were expressed as the signal percentage relative to untreated cells, normalized to the Hsp90-based loading control. Mean ± SEM values from no less than five values across five independent experiments are provided, with significance determined through mixed-effects analysis and Dunnett’s multiple comparisons test, referring to samples treated only with PT (white bar). ( c ) Recombinant Gαi was pre-incubated with either HSF1A, or DMSO as the control, for 30 min at room temperature. For further control, recombinant Gαi was also incubated with buffer exclusively. Subsequently, 100 nM PTS1 and 10 μM biotin-labeled NAD + were added, and incubation was carried out for 30 min at room temperature. The presence of biotin-labeled (ADP-ribosylated) Gαi was identified using streptavidin–peroxidase, and signal intensities were quantified via densitometry. The values are presented as a percentage relative to samples treated solely with PTS1, with mean ± SEM values from no less than four values across four independent experiments. Significance is determined through mixed-effects analysis and Dunnett’s multiple comparisons test, referring to samples treated only with PTS1 (white bar). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Activity Assay, Incubation, Comparison, Control, Solvent, TNKS1 Histone Ribosylation Assay, Labeling, Recombinant

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: HSF1A does not inhibit the binding of PT to cells. ( a ) CHO cells were subjected to a 30 min pre-incubation with either HSF1A or VER, while DMSO was employed as a control, all carried out at 37 °C. Subsequently, cells were exposed to a total of 500 ng/mL PT at a temperature of 4 °C for a duration of 40 min. After thorough washing, the presence of bound PT was detected using Western blotting and a specific PTS1 antibody. Assurance of equivalent loading was validated through Hsp90 detection. The Western blot signals were quantified and then normalized both to the Hsp90 signals and to the samples treated solely with PT (mean ± SEM values), derived from a minimum of four values across four independent experiments. Statistical significance was assessed using mixed-effects analysis and Dunnett’s multiple comparisons test, with the values referring to samples treated exclusively with PT being represented by a white bar. ( b ) A suspension of CHO cells underwent a 30 min pre-incubation with HSF1A or DMSO at 37 °C. Subsequently, the cells were cooled on ice and then exposed to 500 ng/mL PT labeled with 488-dye for a duration of 15 min under ice-cold conditions. After undergoing two wash cycles, the attached PT was identified using flow cytometry. The values presented are indicated as x-fold relative to the control. The dataset comprises 10 values from five separate experiments. Statistical analysis involved a one-way ANOVA test, with the results compared to samples treated exclusively with PT. **** p < 0.0001, ns = not significant.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Binding Assay, Incubation, Control, Western Blot, Derivative Assay, Suspension, Labeling, Flow Cytometry

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: When cells are treated with HSF1A, a reduced amount of PTS1 signal is observed within cells. CHO cells were subjected to a 30 min pre-incubation with HSF1A or VER at 37 °C. For control, cells were left untreated. Following this, PT at a concentration of 100 ng/mL was introduced for a 4 h period. After washing, the cells underwent fixation and permeabilization, followed by staining of PTS1 through incubation with a specific primary antibody, succeeded by a secondary fluorescence-labeled antibody. Nuclei were stained using Hoechst dye, and images were captured in a random fashion using a Keyence fluorescence microscope. The PTS1 signal was quantified from uncropped images (40× objective) and values are shown as percent of PT-only treated cells (mean ± SEM, n = 30 (30 images from three independent experiments). An average of 41.73 ± 1.019 (mean ± SEM) cells were captured per image. Statistical analysis involved a mixed-effects analysis and Dunnett’s multiple comparisons test, with the results compared to samples treated exclusively with PT. **** p < 0.0001. Green = PTS1, blue = nucleus. White squares indicate the magnified areas. Scale bar = 50 µm.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Incubation, Control, Concentration Assay, Staining, Fluorescence, Labeling, Microscopy

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: Interaction of TRiC/CCT subunit CCT5 with PTS1 in cells. A549 cells were pre-incubated with 200 µM HSF1A or the corresponding amount of its solvent, DMSO, for 30 min. Cells were challenged with 100 ng/mL PT for 4 h. Then, cells were carefully washed, fixed, and processed for a fluorescence-based proximity ligation assay (PLA) in accordance with the manufacturer’s guidelines. The cell nuclei were stained with Hoechst (blue). The resulting PLA signals (depicted in white) indicate instances of protein interaction between PTS1 and CCT5. White squares indicate the magnified areas below. The quantification of these signals per cell number is presented in the bar graph. The values are reported as mean ± SEM (n = 40 uncropped images captured in a random fashion using a Keyence fluorescence microscope 40× objective per condition from four independent experiments). An average of 37.09 ± 0.5213 (mean ± SEM) cells were captured per image. Significance levels tested using one-way ANOVA with Dunnett’s multiple comparisons test are indicated by asterisks (**** p ≤ 0.0001, ns denotes non-significant) and were tested against untreated control samples. Scale bar = 25 µm.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Incubation, Solvent, Fluorescence, Proximity Ligation Assay, Staining, Microscopy, Control

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: Schematics of the role of host cell chaperones in pertussis toxin activity. Pertussis toxin (PT) consists of the enzyme subunit PTS1 and the B-subunit pentamer, which facilitates cell binding. The cell binding is followed by endocytosis and retrograde transport of the toxin through the Golgi to the endoplasmic reticulum (ER). In the ER, PTS1 is released from the B-pentamer, unfolded, and transported to the cytosol by the ER-associated degradation pathway. This translocation and subsequent refolding of PTS1 is supported by several chaperones, Hsp90, Hsp70, cyclophilins (Cyps), and FK506 binding proteins (FKBPs), as well as the chaperonin TRiC/CCT complex.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Activity Assay, Binding Assay, Translocation Assay