Journal: Pain

Article Title: Dissecting the role of the CRMP2–neurofibromin complex on pain behaviors

doi: 10.1097/j.pain.0000000000001026

Figure Lengend Snippet: Functional “fingerprinting” of DRG neuronal subclasses after treatment with t-CNRP1. (A) Ca2+-imaging traces of DRG responding to constellation pharmacology agonist or membrane-potential triggers. Each trace represents the response of a different neuron. In a typical experimental trial, the responses of >300 individual neurons were monitored simultaneously. Selected traces are shown. Colored columns indicate the ~15-s application of challenge compounds or high [K+]o; after each application, the free compound or high [K+]o was continuously washed out of the well with room temperature bath solution. The y-axis shows the Fura-2AM fluorescence ratio (F340/F380) for each trace. Raw, unfiltered traces are presented. Triggers used were menthol (400 nM), histamine (50 µM), ATP (10 µM), AITC (200 µM), acetylcholine (1 mM), capsaicin (100 nM), and KCl (90 mM). Dorsal root ganglion neurons were treated with either vehicle (0.01% DMSO) (A) or t-CNRP1 (10 µM) (B) and the neuronal populations analyzed by the constellation pharmacology protocol. All cells were selected based on their response to the depolarizing pulse of KCl. Size of each cell is indicated by the surface area value in each trace. (C) Bar graph showing the overall number of functional DRG neuron classes identified. t-CNRP1 increased the number of functional DRG neuron classes detected. (D) The response of DRG neurons to one or more constellation pharmacology triggers was analyzed. The polar plot indicates the percentage of cells that responded to the indicated number of triggers independent of which compound they responded to. The number 0 corresponds to the proportion of cells that responded to KCl only and no other trigger. After t-CNRP1 treatment, less cells responded to only KCl and increased number of cells responded to one or more constellation pharmacology triggers. (E) Polar plot showing the percent of cells responding to major classes of triggers. Data are from 4 independent experiments with a total n = 1541 for control and n = 1642 for 10 µM t-CNRP1. Bar graphs showing the average peak (F) or area under the curve (AUC) (G) calcium response in the major functional neuronal populations identified in panel (E). Area under the curve was calculated with Prism software using the trapezoid rule. t-CNRP1 did not decrease the peak calcium influx in response to any trigger except Cap for peak or Ach and Cap for AUC (*P < 0.05; Student t test). An AUC for KCl was not calculated in these experiments because KCl was the last trigger (see traces in A and B). (H) Bar graphs showing the average peak response to depolarization for cells showing a response to the indicated trigger independent of which other compound they responded to. T-CNRP1 decreased the peak KCl response for all subclasses except Menth responding DRG neurons. (*P < 0.05; Student t test). Ach, acetylcholine; AITC, allyl isothiocyanate; ATP, adenosine triphosphate; DRG, dorsal root ganglion; Hist, histamine; Menth, menthol; Cap, capsaicin; KCl, potassium chloride.

Article Snippet: Calcium imaging in acutely dissociated dorsal root ganglion neurons Dorsal root ganglion neurons were loaded for 30 minutes at 37˚C with 3 µM Fura-2AM (Cat# F1221, Thermo Fisher, stock solution prepared at 1 mM in DMSO, 0.02% pluronic acid, Cat#P-3000 MP, Life technologies) to follow changes in intracellular calcium ([Ca 2+ ] c ) in a standard bath solution containing 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl 2 , 1.8 mM CaCl 2 , 10 mM Na HEPES, pH 7.4, 5 mM glucose exactly as previously described.

Techniques: Functional Assay, Imaging, Fluorescence, Software

Journal: Cell Death and Differentiation

Article Title: HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

doi: 10.1038/cdd.2015.75

Figure Lengend Snippet: Ser682 phosphorylation regulates SIRT1 deacetylase activity, p53 acetylation and cell death. ( a ) HIPK2 is important for p53 Lys382 acetylation after DNA damage. HIPK2 was depleted in U2OS cells by RNA interference and cells were treated with Adriamycin (0.75 μ g/ml) as indicated. Total cell lysates were analysed by immunoblotting using the indicated antibodies. ( b ) HIPK2 antagonizes SIRT1-mediated deacetylation of p53. Flag-SIRT1, HA-HIPK2, p53 and HA-CBP were expressed in H1299 cells as indicated and total cell lysates were analysed by immunoblotting using the indicated antibodies. GFP expression was used to control the transfection efficiency. Total p53 protein expression levels were adjusted to equal levels to be able to compare p53 acetylation under different conditions. The ratio (amount p53 acK)/(amount p53) was quantified by densitometry using the ImageJ software. ( c ) SIRT1 Ser682 is required for HIPK2-mediated SIRT1 inhibition. H1299 cells were transfected with the indicated constructs and total cell lysates were analysed by immunoblotting using the indicated antibodies. GFP expression was used to control the transfection efficiency. Total p53 protein expression levels were adjusted to equal levels to be able to compare p53 acetylation under different conditions. The ratio (amount p53 acK)/(amount p53) was quantified by densitometry using the ImageJ software. ( d ) SIRT1 Ser682 is important for DNA damage-stimulated p53 acetylation. H1299 cells were transfected with the indicated expression constructs. Twenty-four hours after transfection cells were treated with Adriamycin (0.75 μ g/ml) or left untreated. Total cell lysates were analysed by immunoblotting. GFP expression was used to control the transfection efficiency. Total p53 protein expression levels were adjusted to equal levels to be able to compare p53 acetylation under different conditions. The ratio (amount p53 acK)/(amount p53) was quantified by densitometry using the ImageJ software. ( e ) SIRT1 Ser682 phosphorylation regulates p53-dependent transcription. U2OS cells were transfected with the indicated expression constructs together with a luciferase PUMA-reporter. Firefly reporter activity was normalized to Renilla reporter activity. Data are shown as means±S.D.; n =3; P <0.05, Student's t -test. ( f ) U2OS cells were transfected with the indicated expression constructs and expression of p53 target genes: p21, PUMA, BAX, NOXA and p53AIP1 were analysed by qRT-PCR. mRNA expression levels were normalized to the empty vector control. Data are shown as means±S.D.; n =3; P <0.05, Student's t -test. ( g ) SIRT1 Ser682 phorphorylation potentiates apoptosis. U2OS cells were transfected with the indicated expression constructs. Twenty-four hours after transfection cells were treated with Adriamycin (1 μ g/ml, 48 h) and subsequently analysed by FACS using Annexin V-FITC

Article Snippet: The following antibodies were used: mouse monoclonal SIRT1 7B7 (Novus Biologicals, LLC, Littleton, CO, USA) for immunofluorescence staining and mouse monoclonal SIRT1 Clone 3H10.2 (Millipore, Merck KGaA, Darmstadt, Germany), rabbit polyclonal SIRT1 (M07-131; Millipore, Merck KGaA) for immunoblotting, RPS19BP1 (AROS, ATLAS Antibodies AB, Stockholm, Sweden), PARP and p53 phospho-Ser46 (BD Pharming, Pharmingen, San Diego, CA, USA), p53 DO-1, GFP FL and PML H-238 (Santa Cruz Biotechnologies Inc., Dallas, TX, USA), Flag M2 (Sigma-Aldrich, St Louis, MO, USA), actin C4 (MP Biomedicals, (LLC, Santa Ana, CA, USA)), HA clones 12CA5 and 3F10 (Roche, F. Hoffmann-La Roche Ltd., Basel, Switzerland), p53 phospho-Ser46, SIRT1 (rabbit Ab), SIRT1 phospho-Ser27 (all Cell Signaling Technologies Inc., Danvers, MA, USA), acetyl-Lys373/382 p53 (Millipore, Merck KGaA).

Techniques: Phospho-proteomics, Histone Deacetylase Assay, Activity Assay, Western Blot, Expressing, Control, Transfection, Software, Inhibition, Construct, Luciferase, Quantitative RT-PCR, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

doi: 10.1038/cdd.2015.75

Figure Lengend Snippet: Proposed model for the interplay between HIPK2 and SIRT1 at PML-NBs upon severe genotoxic stress. Our data propose that in response to severe DNA damage SIRT1 and HIPK2 are recruited to PML-NBs by the PML isoform IV. Co-recruitment of both p53 regulatory enzymes facilitates crosstalk between SIRT1 and HIPK2 at the PML-NB. HIPK2 phosphorylates SIRT1, which in turn inhibits SIRT1 activity through dissociation of AROS. Reduced SIRT1 activity enables efficient p53 acetylation, expression of pro-apoptotic p53 target genes and potentiation of the DNA damage-induced cell death response

Article Snippet: The following antibodies were used: mouse monoclonal SIRT1 7B7 (Novus Biologicals, LLC, Littleton, CO, USA) for immunofluorescence staining and mouse monoclonal SIRT1 Clone 3H10.2 (Millipore, Merck KGaA, Darmstadt, Germany), rabbit polyclonal SIRT1 (M07-131; Millipore, Merck KGaA) for immunoblotting, RPS19BP1 (AROS, ATLAS Antibodies AB, Stockholm, Sweden), PARP and p53 phospho-Ser46 (BD Pharming, Pharmingen, San Diego, CA, USA), p53 DO-1, GFP FL and PML H-238 (Santa Cruz Biotechnologies Inc., Dallas, TX, USA), Flag M2 (Sigma-Aldrich, St Louis, MO, USA), actin C4 (MP Biomedicals, (LLC, Santa Ana, CA, USA)), HA clones 12CA5 and 3F10 (Roche, F. Hoffmann-La Roche Ltd., Basel, Switzerland), p53 phospho-Ser46, SIRT1 (rabbit Ab), SIRT1 phospho-Ser27 (all Cell Signaling Technologies Inc., Danvers, MA, USA), acetyl-Lys373/382 p53 (Millipore, Merck KGaA).

Techniques: Activity Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sesamin Enhances Nrf2-Mediated Protective Defense against Oxidative Stress and Inflammation in Colitis via AKT and ERK Activation

doi: 10.1155/2019/2432416

Figure Lengend Snippet: SSM protected Caco-2 cells from H 2 O 2 -induced cytotoxicity via GSH-mediated ROS scavenging. (a) Chemical structure of SSM. (b) Caco-2 cells were treated with 0 μ M SSM (0.1% DMSO) and various concentrations of SSM (5, 10, 20, 40, 80, 160, and 320 μ M) for 8, 16, and 24 h. Cell viability was measured by MTT assay. (c) Caco-2 cells were pretreated with 0 μ M SSM (0.1% DMSO) or 40 μ M SSM for 2, 4, and 8 h and then treated with various concentrations of H 2 O 2 (0, 0.26, 0.6, 0.76, 1.0, 1.26, 1.6, 1.76, and 2.0 mM) for 24 h. Cell viability was measured by MTT assay. (d) Caco-2 cells were pretreated with 0 μ M SSM (0.1% DMSO) and various concentrations of SSM (5, 10, 20, 40, and 80 μ M) for 8 h and then treated with 1.6 mM H 2 O 2 for 24 h. Intracellular ROS were detected using DCFH-DA fluorescence assay. Roseup was used as a positive control (+). Images are displayed at 200x magnification. The quantification of fluorescence was performed with a Flex Station 3 multifunctional microplate reader. Data were expressed as the mean ± SD ( n = 5). ∗∗∗ p < 0.001 versus the control group. # p < 0.05 and ### p < 0.001 versus the H 2 O 2 treatment group. (e, f) Caco-2 cells were treated with 0 μ M SSM (0.1% DMSO) and various concentrations of SSM (10, 20, 40, and 80 μ M) for 8 h. Reduced GSH (GSH) and oxidized GSH (GSSG) were detected using commercial kits. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05 versus the DMSO treatment group. (g) Caco-2 cells were pretreated with 0.1% DMSO (control) or 10 μ M BSO and 0 μ M SSM (0.1% DMSO) or various concentrations of SSM (5, 10, 20, 40, and 80 μ M) for 2 and 8 h, respectively, and then treated with 1.6 mM H 2 O 2 for 24 h. Cell viability was measured by MTT assay. Data were expressed as the mean ± SD ( n = 5). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus the H 2 O 2 treatment group. ## p < 0.01 and ### p < 0.001 versus the control group.

Article Snippet: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from MP Biomedicals (CA, USA).

Techniques: MTT Assay, Fluorescence, Positive Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sesamin Enhances Nrf2-Mediated Protective Defense against Oxidative Stress and Inflammation in Colitis via AKT and ERK Activation

doi: 10.1155/2019/2432416

Figure Lengend Snippet: SSM increased the nuclear translocation of Nrf2 and induced the expressions of cytoprotective genes in Caco-2 cells. (a) The effect of SSM on the protein expressions of Nrf2 and its target genes was analyzed by western blot in Caco-2 cells treated with 0 μ M SSM (0.1% DMSO) and various concentrations of SSM (10, 20, 40, and 80 μ M) for 2 and 8 h. (b–g) Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the DMSO treatment group. (h) Caco-2 cells were transfected with si-control or si-Nrf2 for 48 h. Then, the protein expressions of Nrf2 and its target genes were analyzed by western blot in these cells treated with 0.1% DMSO (-) or 40 μ M SSM (+) for 8 h. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the DMSO treatment group. # p < 0.05 versus the SSM treatment group. (i) Caco-2 cells were transfected with si-control or si-Nrf2 for 48 h. Then, the cells were pretreated with 0 μ M SSM (0.1% DMSO) or 40 μ M SSM for 8 h and 1.6 mM H 2 O 2 for another 24 hours. Cell viability was measured by MTT assay. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the H 2 O 2 treatment group. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the si-control group.

Article Snippet: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from MP Biomedicals (CA, USA).

Techniques: Translocation Assay, Western Blot, Software, Transfection, MTT Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sesamin Enhances Nrf2-Mediated Protective Defense against Oxidative Stress and Inflammation in Colitis via AKT and ERK Activation

doi: 10.1155/2019/2432416

Figure Lengend Snippet: SSM activated Nrf2 signaling through promoting ERK and AKT activation in Caco-2 cells. (a) The effect of SSM on ERK, AKT, P38, PKC, and Keap1 was analyzed by western blot in Caco-2 cells treated with 0 μ M SSM (0.1% DMSO) and various concentrations of SSM (10, 20, 40, and 80 μ M) for 2 and 8 h. (b) Quantitative data were obtained from the densitometric quantification of immunoblots by using ImageJ software. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05 and ∗∗ p < 0.01 versus the DMSO treatment group. (c) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (5 and 10 μ M), or wortmannin (5 and 10 μ M) for 2 h prior to SSM treatment (40 μ M) for 8 h. The protein expressions of AKT and ERK were analyzed by western blot. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05 versus the DMSO treatment group. # p < 0.05 and ### p < 0.001 versus the SSM treatment group. (d) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (5 and 10 μ M), or wortmannin (5 and 10 μ M) for 2 h prior to SSM treatment (40 μ M) for 8 h. The protein expressions of Nrf2 and its target genes were analyzed by western blot. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05 versus the DMSO treatment group. # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the SSM treatment group. (e) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (5 and 10 μ M), or wortmannin (5 and 10 μ M) for 2 h prior to SSM treatment (40 μ M) for 8 h. The nuclear protein expression of Nrf2 was analyzed by western blot. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD ( n = 5). ∗∗∗ p < 0.001 versus the DMSO treatment group. ### p < 0.001 versus the SSM treatment group. (f) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (10 μ M), or wortmannin (10 μ M) for 2 h prior to SSM treatment (40 μ M) for 8 h and then treated with 1.6 mM H 2 O 2 for another 24 h. Images are displayed at 200x magnification using an OLYMPUS IX73 inverted fluorescence phase-contrast microscope. The quantification of fluorescence was done with a Flex Station 3 multifunctional microplate reader. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the control group. (g, h) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (10 μ M), or wortmannin (10 μ M) for 2 h prior to SSM treatment (40 μ M) for 8 h and then treated with 1.6 mM H 2 O 2 for another 24 h. Cell viability was measured by MTT assay. Data were expressed as the mean ± SD ( n = 5). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the control group.

Article Snippet: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from MP Biomedicals (CA, USA).

Techniques: Activation Assay, Western Blot, Software, Expressing, Fluorescence, Microscopy, MTT Assay