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Figure Lengend Snippet: a End-point activity assay of the wild-type sGC samples in the presence or absence of 20 mM NS2028, 20 mM cinaciguat or 100 mM DEA. NS2028 oxidizes the ferrous haem in sGC to ferric haem. Mean ± s.d., n = 3 biologically independent reactions. Source data are provided as a . b End-point activity assay of the haem-free sGC samples with or without 20 mM cinaciguat or 100 mM DEA as indicated. Mean ± s.d., n = 3 biologically independent reactions. Source data are provided as a . c Density at the cinaciguat binding pocket in the CI state. Cinaciguat is shown as magenta sticks. d The interactions between cinaciguat and sGC in the CI state. A 45° rotated view compared to ( c ). e Cryo-EM map of sGC in the cinaciguat bound inactive (CI) state. The density of cinaciguat is shown in magenta. f Structural comparison of sGC in the CI state (colored) and haem-oxidized state (gray) by aligning the β1 H-NOX domain. g A 90° rotated zoom-in view of the β1 H-NOX domain boxed in f (black rectangle). The side chains of H105, Y112, and F4 are shown as sticks. h A 90° rotated view of catalytic module boxed in f (red rectangle). The angle between the αQ helices is shown.

Article Snippet: For the cinaciguat-bound sGC, two approaches were used for the sample preparation: in the first approach, the wild type sGC with ferrous haem were incubated with 200 μM NS2028 and 200 μM cinaciguat (TargetMol) at room temperature overnight, and then 1 mM GMPCPP, 5 mM MgCl 2 , 0.5 mM FOM were added; in the second approach, the purified haem-free sGC was concentrated to A 280 = 4.5, and then 200 μM cinaciguat, 1 mM GMPCPP, 5 mM MgCl 2 , 0.5 mM FOM were added at 4 °C.

Techniques: Activity Assay, Binding Assay, Cryo-EM Sample Prep, Comparison

Journal:

Article Title:

doi:

Figure Lengend Snippet: a Cryo-EM map of sGC in the cinaciguat-bound activated state (CA) state. The density of cinaciguat is shown in green. b The cut-open view of ( a ). c Density at the cinaciguat-binding site in CA state. Cinaciguat is shown as dark green sticks. d The interactions between cinaciguat and sGC. A 45° rotated view compared to ( b ). e Structure comparison of sGC β1 H-NOX in the CA state (colored) and the NO-activated state (gray). f Structural comparison of cinaciguat in the Nostoc H-NOX domain (PDB ID: 3L6J, yellow), CI state (magenta) and CA state (green). The core of cinaciguat is indicated by dashes. g The interactions between the terminal benzyl group of cinaciguat and F4 and Y112 of sGC in the CI state. h The interactions between the terminal benzyl group of cinaciguat and F4 and Y112 of sGC in the CA state. i The interaction between the terminal benzyl group of cinaciguat and L4 and F112 of the Nostoc H-NOX domain (PDB ID: 3L6J). j End-point activity assay of the various sGC mutants in the presence or absence of 20 mM NS2028 and 20 mM cinaciguat as indicated. Mean ± s.d., n = 3 biologically independent reactions. Source data are provided as a .

Article Snippet: For the cinaciguat-bound sGC, two approaches were used for the sample preparation: in the first approach, the wild type sGC with ferrous haem were incubated with 200 μM NS2028 and 200 μM cinaciguat (TargetMol) at room temperature overnight, and then 1 mM GMPCPP, 5 mM MgCl 2 , 0.5 mM FOM were added; in the second approach, the purified haem-free sGC was concentrated to A 280 = 4.5, and then 200 μM cinaciguat, 1 mM GMPCPP, 5 mM MgCl 2 , 0.5 mM FOM were added at 4 °C.

Techniques: Cryo-EM Sample Prep, Binding Assay, Comparison, Activity Assay

Journal: Journal of cell communication and signaling

Article Title: Mutant p53 gain-of-function stimulates canonical Wnt signaling via PI3K/AKT pathway in colon cancer.

doi: 10.1007/s12079-023-00793-4

Figure Lengend Snippet: Fig. 3 Mutant p53 knockdown in the SW480 cell line decreases canonical Wnt pathway. A Comparation of non-phospho- rylated β-catenin (active) and p53 protein levels measured in different colon cancer cell lines with different TP53 status, RKO (wild-type p53), SW480 (R273H p53), and SW620 (R273H p53). Densitometric analysis of immunoblots of p53 and active β-catenin was performed using GAPDH as a loading control. B Representa- tive immunoblots and graph of p53 protein in SW480 under different doses of siRNA p53 (25–100 nM); the scramble condition was used as a control (100 nM). C Luciferase activity measured with the TOP/FOP system in both scramble and siRNA p53 (100 nM) condi- tions. The relative units were normalized to Renilla activity. D Representative immunoblot and graph of c-myc normalized to GAPDH. E. Representa- tive colonies and relative area quantification corresponding to scramble and shp53 conditions of SW480-transduced cells. Graphs represent densito- metric analysis from at least three independent experiments (means ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The PI3K/AKT inhibitor wortmannin was obtained from Sigma (Cat. No. 19545-26-7), and the p53 Activator III compound RITA was from Santa Cruz Biotechnology (Cat. No. sc-202743).

Techniques: Mutagenesis, Knockdown, Western Blot, Control, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

doi: 10.1371/journal.pone.0023596

Figure Lengend Snippet: cGMP reporter cells were cotransfected with WT α 1 sGC and TC4-WT β 1 sGC and labelled with FlAsH. The cells were then incubated with 0.1, 1 or 10 µM BAY 58-2667 (A) 1, 30 or 100 µM rotenone (B) or 1, 10 or 100 µM NS 2028 (C) for 90 min. Fluorescence intensity of single cells was monitored in a time series of 120 laser scans and is expressed as % of start value which was obtained before application of the test compounds. Data are means ± S.E.M. from 9–28 single cells assayed on different days. * p <0.05; *** p <0.005: Student's t -test compared to control. Representative traces of the fluorescence measurement as % of start value are given in the right part of the figure.

Article Snippet: ODQ (1H-(1,2,4)-oxadiazole(4,3- a )quinoxalin-1-one) was purchased from Tocris Bioscience (Avonmouth, UK) and NS 2028 (4 H -8-Bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one) was purchased from Axxora GmbH (Loerrach, Germany).

Techniques: Incubation, Fluorescence

Journal: PLoS ONE

Article Title: Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

doi: 10.1371/journal.pone.0023596

Figure Lengend Snippet: Effects of 100 µM NS 2028 on fluorescence intensity of FlAsH labelled haem- free TC4-Y135A/R139A sGC (A) and ReAsH labelled TC4-WT sGC (B) after 90 min incubation.

Article Snippet: ODQ (1H-(1,2,4)-oxadiazole(4,3- a )quinoxalin-1-one) was purchased from Tocris Bioscience (Avonmouth, UK) and NS 2028 (4 H -8-Bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one) was purchased from Axxora GmbH (Loerrach, Germany).

Techniques: Fluorescence, Incubation

Journal: PLoS ONE

Article Title: Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

doi: 10.1371/journal.pone.0023596

Figure Lengend Snippet: cGMP reporter cells expressing TC4-WT sGC were pre-incubated with 100 µM NS 2028 or rotenone and then incubated with increasing concentrations of BAY 58-2667, BAY 41-2272 alone or in combination with 10 µM ODQ or 10 nM DEA/NO (NO), respectively. Enzyme activity is expressed as x-fold stimulation compared to pre-treated but not stimulated control. Data are means ± S.E.M. from 3–10 independent experiments, performed in duplicate. A basal activity of 7747 relative light units was measured. A) is identical to .

Article Snippet: ODQ (1H-(1,2,4)-oxadiazole(4,3- a )quinoxalin-1-one) was purchased from Tocris Bioscience (Avonmouth, UK) and NS 2028 (4 H -8-Bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one) was purchased from Axxora GmbH (Loerrach, Germany).

Techniques: Expressing, Incubation, Activity Assay

Journal: PLoS ONE

Article Title: Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

doi: 10.1371/journal.pone.0023596

Figure Lengend Snippet: Effects of 90 min pre-treatment with 100 µM NS 2028 or rotenone on TC4-WT sGC protein levels.

Article Snippet: ODQ (1H-(1,2,4)-oxadiazole(4,3- a )quinoxalin-1-one) was purchased from Tocris Bioscience (Avonmouth, UK) and NS 2028 (4 H -8-Bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one) was purchased from Axxora GmbH (Loerrach, Germany).

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