Journal: bioRxiv

Article Title: Expanding the ligandable proteome by paralog hopping with covalent probes

doi: 10.1101/2024.01.18.576274

Figure Lengend Snippet: a , Structures of two inhibitors of the CCNE1:CDK2 complex – I-125A and I-198 – reported in a patent . b , ADP- Glo data showing effects of the indicated compounds on the activity of a purified N112C-CCNE1:CDK2 complex as measured by phosphorylation of the C-terminal peptide of RB1 (left) or the histone H1 protein (right). The N112C-CCNE1:CDK2 complex was pre-treated with compounds for 2 h (37 °C) before initiating phosphorylation reactions by addition of substrate. CI, confident intervals. Data represent average values ± s.e.m., n = 3. c , Gel-ABPP data showing concentration-dependent partial blockade of YZ-01-A engagement of N112-CCNE1 by I-125A and I-198, but not dinaciclib. Assays were performed in lysates from transfected HEK293T cells co-expressing NLuc-N112C-CCNE1 or NLuc-WT-CCNE1 and CDK2 that were treated with the indicated concentration ranges of compounds followed by YZ-01-A (10 µM, 90 min), anti-FLAG immunoprecipitation, and processing for gel-ABPP. d , Quantification of gel-ABPP data shown in c . Data represent average values ± s.e.m., n = 3. e , NanoBRET-ABPP data showing concentration-dependent partial blockade of YZ-01-A engagement of N112-CCNE1 by I-125A and I-198. Samples were treated with compounds as described in c prior to NanoBRET measurement. Data represent average values ± s.e.m., n = 3. f , Quantification of gel- and NanoBRET-ABPP data showing that dinaciclib does not substantially affect YZ-01-A engagement of N112C-CCNE1. Data represent average values ± s.e.m., n = 3.

Article Snippet: Briefly, different CCNE: CDK2 purified complexes at 4 ng µL - were mixed 1:1 with 2x compound stock (Both are made in kinase reaction buffer (40 mM Tris [pH = 7.6], 20 mM MgCl2, 0.1 mg mL - BSA and 50 µM DTT) and incubated at 37 ℃ for 1 h. Then 6 ng compound treated CCNE: CDK2 complexes (3 µL) were mixed with 2µL ATP/ CDK2 substrate mix to make final concentration of 150 µM ATP and 0.5 µg substrate per reaction (Substrate used is either histone H1(Sigma-Aldrich, 14-155) or RB1 C-terminal peptide (MyBioSource, MBS143254)), then incubated at room temperature for 1 h. Subsequently, 5 µL ADP-Glo reagent was added and incubated for 40 min at room temperature, followed with 10 µL kinase glo reagent and another 30-min incubation.

Techniques: Activity Assay, Purification, Concentration Assay, Transfection, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Expanding the ligandable proteome by paralog hopping with covalent probes

doi: 10.1101/2024.01.18.576274

Figure Lengend Snippet: a, b , ADP-Glo data showing effects of the indicated compounds on the activity of a purified WT-CCNE1:CDK2 ( a ) or WT-CCNE2:CDK2 ( b ) complexes as measured by phosphorylation of the C-terminal peptide of RB1 (left) or the histone H1 protein (right). The WT-CCNE:CDK2 complexes were pre-treated with compounds for 2 h (37 °C) before initiating phosphorylation reactions by addition of substrate. CI, confident intervals. Data represent average values ± s.e.m., n = 3. c, Gel-ABPP data showing concentration-dependent blockade of YZ-01-A engagement of N112C-CCNE1 following incubation with I-125A or I-198 for 30 min or 90 min. Experiment was performed as described in . Data are from a single experiment. d , Gel-ABPP data showing that residual YZ-01-A reactivity with N112C-CCNE1 in presence of 1 µM of I-125A or I-198 is stereoselective (greater than signals generated with YZ-01-B) and site-specific (greater than signals generated with WT-CCNE1). Experiments were performed as described in . Data represent average values ± s.e.m., n = 2. e , NanoBRET data showing concentration-dependent blockade of active site-directed tracer K-10 (0.2 µM) binding to N112C-CCNE1:CDK2-NLuc (left) or WT-CCNE1:CDK2-NLuc complexes by dinaciclib, but not I-125A. NanoBRET assays were performed in lysates of HEK293T cells recombinantly expressing the CCNE1:CDK2-NLuc complexes. Data represent average values ± s.e.m., n = 3.

Article Snippet: Briefly, different CCNE: CDK2 purified complexes at 4 ng µL - were mixed 1:1 with 2x compound stock (Both are made in kinase reaction buffer (40 mM Tris [pH = 7.6], 20 mM MgCl2, 0.1 mg mL - BSA and 50 µM DTT) and incubated at 37 ℃ for 1 h. Then 6 ng compound treated CCNE: CDK2 complexes (3 µL) were mixed with 2µL ATP/ CDK2 substrate mix to make final concentration of 150 µM ATP and 0.5 µg substrate per reaction (Substrate used is either histone H1(Sigma-Aldrich, 14-155) or RB1 C-terminal peptide (MyBioSource, MBS143254)), then incubated at room temperature for 1 h. Subsequently, 5 µL ADP-Glo reagent was added and incubated for 40 min at room temperature, followed with 10 µL kinase glo reagent and another 30-min incubation.

Techniques: Activity Assay, Purification, Concentration Assay, Incubation, Generated, Binding Assay, Expressing

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 1. Rb1 protected the brain against oxygen and glucose deprivation and reoxygenation injury (OGD/R). Slices of the hippocampus were exposed to oxygen and glucose deprivation for 4 h, followed by reoxygenation for 1 h. A. Representative pictures and quantification of TUNEL staining (one of three independent exper iments, scale bar 50 μm); B. LDH released in the medium (n = 5); C. Representative pictures and quantification of intracellular ROS production (one of three in dependent experiments, scale bar 50 μm); D. 8-OHdG contents (n = 5); E. Immunoblot of glutamine synthetase (GS, n = 5); F. glutamate released in the medium (n = 5). G. Representative pictures and quantification of GFAP staining (one of three independent experiments, scale bar 50 μm). Data are presented as mean ± SD. *p < 0.05 vs.the OGD/R only treatment; #p < 0.05 vs. indicated treatments. OGD/R, oxygen glucose deprivation/reperfusion; Rb1, ginsenoside Rb1; GS, glutamine synthetase; GFAP, glial fibrillary acidic protein.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: TUNEL Assay, Staining, Western Blot

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 2. Rb1 inhibited astrocyte reactivity. A. Intracellular ROS production (n = 5); B. Intracellular calcium signal (n = 6); C. Representative pictures and quanti fication of GFAP staining in astrocytes (one of three independent experiments, scale bar 10 μm); D. ROS production in astrocytes exposed to succinate and oligomycin A (n = 6); E. Intracellular calcium signal in astrocytes (one of three independent experiments, scale bar 10 μm); F. Representative pictures and quantification of GFAP staining in astrocytes exposed to succinate and oligomycin A (one of three independent experiments, scale bar 10 μm). Data are presented as mean ± SD. *p < 0.05 vs. the OGD/R only treatment or succinate plus oligomycin A treatment; #p < 0.05 vs. indicated treatments. OGD/R, oxygen glucose deprivation/reperfusion; Rb1, ginsenoside Rb1; NAC, n-acetylcysteine; GFAP, glial fibrillary acidic protein.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: Staining

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 3. Rb1 inhibited mitochondrial complex I to prevent astrocytes reactivity. A. Representative pictures and quantification of mitochondrial ROS production in response to oxygen and glucose deprivation and reoxygenation (OGD/R)(one of three independent experiments, scale bar 10 μm); B, C. Mitochondrial complex I activity in astrocytes in the presence or absence of succinate and oligomycin A (n = 5); D. Immunoblot for the efficiency of Ndi1 transfection (n = 5); E, F. Intracellular ROS production and calcium in Ndi1-expressed astrocytes (n = 5); G. Representative pictures and quantification of GFAP staining in Ndi1-expressed astrocytes (one of three independent experiments, scale bar 10 μm). Data are presented as mean ± SD. *p < 0.05 vs. the untreated control or succinate plus oli gomycin A treatment; #p < 0.05 vs. indicated treatments; ns: no significant difference. OGD/R, oxygen glucose deprivation/reperfusion; Rb1, ginsenoside Rb1.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: Activity Assay, Western Blot, Transfection, Staining, Control

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 4. Rb1 protected astrocyte function. Astrocytes were exposed to oxygen and glucose deprivation for 4 h, followed by reoxygenation for 1 h (OGD/R). A. Gene expression of Slc7a11 and Gclc (n = 5); B. Intracellular GSH and NADPH contents (n = 6); C. Gene expression of neurotropic factors (n = 6); D. Gene expression of phagocytic factors, Mertk and Megf10 (n = 5); E. Glutamate contents in the medium (n = 6); F. Immunoblot of glutamine synthetase (GS, n = 5). Data are presented as mean ± SD. *p < 0.05 vs. the OGD/R only treatment; #p < 0.05 vs. indicated treatments. OGD/R, oxygen glucose deprivation/reperfusion; Rb1, ginsenoside Rb1; NAC, n-acetylcysteine; GS, glutamine synthetase.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: Gene Expression, Western Blot

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 6. Rb1 treatment in astrocytes conferred neuroprotection. After exposure to oxygen and glucose deprivation for 4 h, neurons were re-cultured in conditioned astrocyte-derived medium for 18 h with reoxygenation. A. LDH release from neurons (n = 6); B. Gene expression of Pdk2 in neurons (n = 5); C. Oxygen consumption rate (OCR) in neurons (n = 6); D, E. Representative pictures and quantification of mitochondrial signal and membrane potential in neurons (one of three independent experiments, scale bar 10 μm); Data are presented as mean ± SD. *p < 0.05 vs. the OGD/R only treatment; #p < 0.05 vs. indicated treatments; ns: no significant difference. OGD/R, oxygen glucose deprivation/reperfusion; Rb1, ginsenoside Rb1; Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone; Anti A, antimycin A; TMRE, tetramethylrhodamine ethyl ester.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: Cell Culture, Derivative Assay, Gene Expression, Membrane

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 7. The neuroprotective effects of Rb1 in mouse stroke model. A. Gene and protein expression of Cd38 knockdown in cerebral ventricles (n = 6); B. Brain infarct size and quantification of infarction area (one of four independent experiments); C. Representative pictures and quantification of immunofluorescence staining of GFAP in the brain (one of three independent experiments, scale bar 100 μm). D. Representative pictures and quantification of ROS production in the brain (one of three independent experiments, scale bar 100 μm); E. Representative pictures and quantification of immunofluorescence staining of Iba 1 in the brain (one of three independent experiments, scale bar 100 μm); F. Representative pictures and quantification of TUNEL staining in the brain (one of three independent experiments, scale bar 100 μm). Data are presented as mean ± SD. *p < 0.05 vs. the photothrombosis only treatment; #p < 0.05 vs. indicated treatments. Rb1, ginsenoside Rb1; GFAP, glial fibrillary acidic protein; Iba 1, ionized calcium binding adaptor molecule 1.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: Expressing, Knockdown, Immunofluorescence, Staining, TUNEL Assay, Binding Assay

Journal: Redox biology

Article Title: Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke.

doi: 10.1016/j.redox.2022.102363

Figure Lengend Snippet: Fig. 8. Schematic representation of the neuroprotective effect of Rb1. Ischemic stroke affects astrocytes and impairs mitochondrial transfer. Transient inhibition of mitochondrial complex I by ginsenoside Rb1 blocks reverse electron transfer-driven ROS production from complex I and prevents astrocyte activation and mito chondrial dysfunction, resultantly facilitating mitochondrial function, functional mitochondrial transfer, and neuronal survival.

Article Snippet: To mimic ischemia and reperfusion injury during stroke, cells were Abbreviations GFAP glial fibrillary acidic protein GS glutamine synthetase LDH lactate dehydrogenase NAC N-acetyl-L-cysteine OCR oxygen consumption rate OGD/R oxygen-glucose deprivation and reperfusion Rb1 ginsenoside Rb1 RET reverse electron transfer ROS reactive oxygen species X.-C. Ni et al. Redox Biology 54 (2022) 102363 pretreated with indicated agents at given concentrations and then subjected to hypoxia in a hypoxia incubator chamber in which O2 was replaced by N2 (37 ◦C, 1% O2, 5% CO2, and 94% N2) for 4 h in glucosefree DMEM (Gibco, 11966), followed by 1 h reoxygenation with glucose supplement.

Techniques: Inhibition, Activation Assay, Functional Assay