Journal: Stem Cell Reports

Article Title: Chemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells

doi: 10.1016/j.stemcr.2024.11.012

Figure Lengend Snippet: Development of a fully synthetic system for highly efficient DE induction from hPSCs (A) Immunofluorescence analysis of DE markers SOX17 and FOXA2 on hESCs differentiated in E5 or B27 supplement-containing culture medium from 3 days as illustrated by the upper schematic ( n = 6 biologically independent experiments). CH, CHIR99021. Scale bars, 50 μm. (B) Percentage of SOX17 + /FOXA2 + DE, cell yields, and survival rate of cells under the indicted conditions at D3 ( n = 6 biologically independent experiments). A minus mark demonstrates withdrawal of the indicated component. I, insulin; T, transferrin; S, sodium selenite; V, vitamin C. (C and D) Representative (C) and quantitative (D) immunofluorescence analysis of SOX17 and FOXA2 on hESCs differentiated on the indicated matrix substrates for 3 days ( n = 6 biologically independent experiments). Scale bars, 50 μm. (E and F) Workflow (E) and high-throughput small screening results (F) for DE inducers in conjunction with 3C. Activin-A and DMSO serve as positive or negative controls, respectively. Chemical structure of the leading hit LDN-193189 is shown. (G) Quantitative reverse-transcription PCR (RT-qPCR) analyses of the expression of key DE transcripts ( n = 9 biologically independent experiments). (H) Immunofluorescence analysis of DE markers SOX17, FOXA2, GATA4, and GATA6 with quantification of the SOX17 + /FOXA2 + and GATA4 + /GATA6 + rations on D3 DE induced by 4C illustrated by the upper schematic ( n = 6 biologically independent experiments). Scale bars, 50 μm. (I) Flow cytometric analyses of the DE marker protein CXCR4 in 4C-DE at D3 ( n = 6 biologically independent experiments). Data are represented as mean ± SE. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; n.s., no significant.

Article Snippet: CHIR99021 , TargetMol T2301 , 3 μM 49 , , 3 μM 49 , .

Techniques: Immunofluorescence, High Throughput Screening Assay, Reverse Transcription, Quantitative RT-PCR, Expressing, Marker

Journal: Stem Cell Reports

Article Title: Chemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells

doi: 10.1016/j.stemcr.2024.11.012

Figure Lengend Snippet: Formulation and price of components of medium for AA- or 4C-based method

Article Snippet: CHIR99021 , TargetMol T2301 , 3 μM 49 , , 3 μM 49 , .

Techniques: Formulation, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase.

doi: 10.1074/jbc.273.26.16415

Figure Lengend Snippet: FIG. 1. Inhibition of MAPKAPK 2 activity by the specific p38 inhibitor SB202190. Jurkat cells were preincubated with or without different concentration of SB202190 for 30 min and then treated with anti-Fas mAb (100 ng/ml) (A) or left alone (B), as indicated. After 2 h, cells were harvested, and endogenous MAPKAPK 2 was immunopre- cipitated with anti-MAPKAPK 2 (Upstate Biotechnology Inc.) from cell extracts (150 mg). The activity of MAPKAPK 2 was measured in im- mune complex kinase assays with GST-hsp27 (5 mg) as a substrate.

Article Snippet: After infection, cells were cultured in serum-free medium and treated with the specific p38 inhibitor SB202190, anti-Fas mAb (Ch11, Santa Cruz) (100 ng/ml), or UV irra- diation (40 J/m2) for various periods of time as indicated in the figure legends.

Techniques: Inhibition, Activity Assay, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase.

doi: 10.1074/jbc.273.26.16415

Figure Lengend Snippet: FIG. 2. The specific p38 inhibitor SB202190 induces cell death in Jurkat cells. A, time course of cell death induced by SB202190. Jurkat cells were serum-starved for 24 h and then treated with the specific p38 inhibitor SB202190, the inactive inhibitor SB202474 (50 mM each), or the specific MEK1 inhibitor PD098059 (100 mM) or left untreated, for various period of times as indicated. The cell viability was monitored by trypan blue exclusion. B, SB202190 induces cell death in a dose-dependent manner. Jurkat cells were serum-starved and then treated with different concentration of SB202190 or PD098059 or left untreated for 24 h. The cell death was measured by propidium iodide staining. C and D, the same as A and B, except that the cells were HeLa cells.

Article Snippet: After infection, cells were cultured in serum-free medium and treated with the specific p38 inhibitor SB202190, anti-Fas mAb (Ch11, Santa Cruz) (100 ng/ml), or UV irra- diation (40 J/m2) for various periods of time as indicated in the figure legends.

Techniques: Concentration Assay, Staining

Journal: The Journal of biological chemistry

Article Title: Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase.

doi: 10.1074/jbc.273.26.16415

Figure Lengend Snippet: FIG. 3. SB202190-induced cell death is programmed cell death. Jurkat cells were treated with SB202190 (50 mM) or PD098059 (100 mM) or left untreated, as indicated. After 24 h, cells were collected. A, the nucleus condensation of the cells was analyzed by staining with H33258. B, the DNA fragmentation was measured by less than 2N DNA.

Article Snippet: After infection, cells were cultured in serum-free medium and treated with the specific p38 inhibitor SB202190, anti-Fas mAb (Ch11, Santa Cruz) (100 ng/ml), or UV irra- diation (40 J/m2) for various periods of time as indicated in the figure legends.

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase.

doi: 10.1074/jbc.273.26.16415

Figure Lengend Snippet: FIG. 4. SB202190-induced cell death requires activation of CPP32-like caspase(s) and is blocked by expression of bcl-2. Jurkat cells stably transfected with bcl-2 expression vector (Jurkat/ bcl-2) or empty neomycin gene (Jurkat/neo) were treated with SB202190 (50 mM) or PD098059 (50 mM) in the presence or absence of zVAD (50 mM) as indicated. After 24 h, cells were harvested, and the cell death was analyzed. A, the activity of CPP32-like caspases of cell extracts (20 mg) was measured using DEVD-AMC peptide as a sub- strate. B, the DNA fragmentation was measured by less than 2N DNA.

Article Snippet: After infection, cells were cultured in serum-free medium and treated with the specific p38 inhibitor SB202190, anti-Fas mAb (Ch11, Santa Cruz) (100 ng/ml), or UV irra- diation (40 J/m2) for various periods of time as indicated in the figure legends.

Techniques: Activation Assay, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase.

doi: 10.1074/jbc.273.26.16415

Figure Lengend Snippet: FIG. 5. SB202190 potentiated cell death induced by anti-Fas mAb and UV irradiation. A, B, and C, Jurkat cells were treated with different amounts of anti-Fas mAb for various period of times in the presence or absence of SB202190 (50 mM) or left untreated, as indicated. D, HeLa cells were irradiated with UV (40 J/m2) and then incubated at 37 °C for various period of times in the presence or absence of SB202190 (50 mM). The death of cells was analyzed by flow cytometry, using propidium iodide as an indicator.

Article Snippet: After infection, cells were cultured in serum-free medium and treated with the specific p38 inhibitor SB202190, anti-Fas mAb (Ch11, Santa Cruz) (100 ng/ml), or UV irra- diation (40 J/m2) for various periods of time as indicated in the figure legends.

Techniques: Irradiation, Incubation, Flow Cytometry

Journal: bioRxiv

Article Title: Characterization of the p38α MAPK allosteric inhibition by a single chain Fv antibody

doi: 10.64898/2026.01.06.697868

Figure Lengend Snippet: (A-B) The in vitro kinase inhibition potency of p38α inhibitors was measured in ADP-Glo assays using as substrate a synthetic peptide (IPTTPITTTYFFFKKK) that contains Ser and Thr phosphorylation sites of kinase proteins. After preincubation of the inhibitors with p38α for 20 min, reactions were started by adding ATP, and stopped after 1h incubation at room temperature. (A) Dose-response inhibition of p38α activity by the scFv G9 and SB 202190 (n=3 independent experiments). (B) Combination of scFv G9 and SB 202190. The black line represents the percentage of p38α kinase activity calculated according to the Bliss’ model assuming the independence of the two inhibitors. (C) Association curves for the binding of SKF-86002 to p38α, determined by real-time measurements of the ATP-competitive inhibitor SKF-86002 fluorescence emission at 420 nm. The excitation wavelength was 340 nm. SKF-86002 was first added to a p38α solution at time 30 sec and then SB 202190, scFv G9 or scFv 13R4 (irrelevant) were added at time 90 sec. The decrease in fluorescence signal corresponds to SKF86002 displacement from the p38α ATP binding site. The concentrations used are: p38α 200 nM; SKF86002 200 nM; SB202190 2 µM; scFv 1 µM. For each compound, n=8. Time-resolved fluorescence was measured using the FLEXstation 3 reader.

Article Snippet: Except when specified for competition experiments, P-p38α (30 nM) or P-p38β (90 nM) were mixed with the peptide substrate IPTTPITTTYFFFKKK (SignalChem) (0.2 mg/mL) and different concentrations of the inhibitor, SB 202190 or scFv G9, in kinase buffer (40 mM Tris pH7.5, 20 mM MgCl 2 , 50 μM DTT, 0.1 mg/mL BSA).

Techniques: In Vitro, Inhibition, Phospho-proteomics, Incubation, Activity Assay, Binding Assay, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Mitogen-activated Protein Kinase Phosphatase-1 Represses c-Jun NH2-terminal Kinase-mediated Apoptosis via NF-κB Regulation

doi: 10.1074/jbc.m802229200

Figure Lengend Snippet: FIGURE 6. Inhibition of JNK, but not p38 and ERK, inhibited radiation-induced m decrease. WT and MKP-1/ MEFs were treated with or without JNK inhibitor SP600125 (2 M), ERK inhibitor U0126 (20 M), p38 inhibitor SB202190 (1 M), or Me2SO (solvent negative control) for 60 min followed by radiation with 0 or 10 Gy of -ray. Protein activity (A) and m (B) were determined at 24 h post-radiation (the basal m of WT without radiation in the presence of Me2SO was set as 1; means S.E., n 3; **, p 0.01). WT and MKP-1/ MEFs were transfected with 100 nM of mouse JNK1/2 siRNA for 24 h and JNK protein levels (C) and m (D) were measured at 24 h post-radiation with 10 Gy of -ray (means S.E., n 3; **, p 0.01).

Article Snippet: JNK inhibitor SP600125 was purchased from Calbiochem (Gibbstown, NJ); ERK inhibitor U0126 was purchased from VWR International (West Chester, PA); protease inhibitors, p38 inhibitor SB 202190, IKK- inhibitor IMD-0354, the protein synthesis inhibitor cycloheximide (CHX), and trypan blue solutionwere purchased fromSigma; proteasome inhibitor was purchased from Peptides International Inc. (Louisville, KY); caspase 9 and GST-c-Jun were purchased from Cell Signaling (Danvers, MA); [ -32P]ATP (6000 Ci/mmol) was purchased from Amersham Biosciences; the JC-1 was purchased from Invitrogen; the TNF and IL-1 were from PeproTech Inc. (Rocky Hill, NJ); and the caspase inhibitor Ac-Val-Ala-AspCMK was purchased form Anaspec Inc. (San Jose, CA).

Techniques: Inhibition, Solvent, Negative Control, Activity Assay, Transfection