kn62  (Santa Cruz Biotechnology)

Journal: Journal of Molecular Cell Biology

Article Title: Wnt/β-catenin pathway induces cardiac dysfunction via AKAP6-mediated RyR2 phosphorylation and sarcoplasmic reticulum calcium leakage

doi: 10.1093/jmcb/mjaf002

Figure Lengend Snippet: Activation of the Wnt/β-catenin pathway disturbs calcium homeostasis in HL-1 cells. ( A ) Confocal images of HL-1 cells treated with or without Wnt3a for 6 h. Fluo4-AM dye and R-CEPIA1er are used to indicate cytosol and SR calcium signals, respectively. Fluorescence intensity (F.I.) was calculated to represent the calcium level. ( B – E ) Cytosol and SR calcium levels in HL-1 cells treated with the indicated compounds for 6 h. Wnt3a: 500 ng/ml; LiCl: 20 mM; CHIR: 5 μM; SFRP2: 10 μg/ml; XAV939: 1 μM; LF3: 1 μM; Wnt5a: 500 ng/ml; Box5: 10 μM; KN62: 1 μM. Tg (100 nM) was used as a positive control. ( F and G ) HL-1 cells preincubated with SFRP2 (10 μg/ml) for 15 min could eliminate the effects of canonical Wnt activators on cytosol and SR calcium levels. Error bars indicate SD ( n = 6).

Article Snippet: Then, various compounds, including SFRP2 (MCE, HY- P77835 ), XAV-939 (MCE, HY-15147), LF3 (MCE, HY-101486), LiCl (Sigma, 656984), thapsigargin (MCE, HY-13433), KN62 (MCE, HY-13290), Box5 (MCE, HY-123071), and cycloheximide (MCE, HY-12320), at different concentrations were added to the cells.

Techniques: Activation Assay, Fluorescence, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: The Cannabinoid CB 1 Receptor Inverse Agonist/Antagonist SR141716A Activates the Adenylate Cyclase/PKA Signaling Pathway Among Other Intracellular Emetic Signals to Evoke Vomiting in Least Shrews ( Cryptotis parva )

doi: 10.3390/ijms26209884

Figure Lengend Snippet: Effects of modulators of cell membrane Ca 2+ channels on CB 1 receptor inverse agonist/antagonist SR141716A (20 mg/kg, i.p.)-induced vomiting. Different groups of least shrews were given an injection of either the corresponding vehicle or varying doses of one of the following: (1) CaMKII inhibitor KN62 (i.p.); (2) the L-type Ca 2+ channel (LTCC) inhibitors nifedipine (s.c.) and amlodipine (i.p.); (3) store-operated Ca 2+ entry blockers YM 58483 (i.p.) and MRS 1845 (i.p.); and (4) the TRPV1R agonist resiniferatoxin (RTX) (s.c.), 30 min prior to SR141716A (20 mg/kg, i.p.) injection. Emetic parameters were recorded for the next 30 min. The frequency of emesis ( A , C , E , G , I , K ) was analyzed using Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and presented as mean ± SEM. The percentage of shrews vomiting ( B , D , F , H , J , L ) was analyzed using the chi-square test and presented as mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 0 mg/kg. The number of animals in each group is presented at the top of the corresponding column.

Article Snippet: The following drugs were used for the present studies: SR141716A, H-89, U0126, PD98059, AR-A014418, SB216763, LY294002, U73122, GF109203X, KN62, amlodipine, YM-58483, MRS-1845, and RTX were purchased from Tocris (Minneapolis, MN, USA); nifedipine and sulpiride were purchased from Sigma-Aldrich (St. Louis, MO, USA); dantrolene and 2-APB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and palonosetron was kindly provided by Helsinn Health Care (Lugano, Switzerland).

Techniques: Membrane, Injection

Journal: International Journal of Molecular Sciences

Article Title: The Cannabinoid CB 1 Receptor Inverse Agonist/Antagonist SR141716A Activates the Adenylate Cyclase/PKA Signaling Pathway Among Other Intracellular Emetic Signals to Evoke Vomiting in Least Shrews ( Cryptotis parva )

doi: 10.3390/ijms26209884

Figure Lengend Snippet: Effects of modulators of cell membrane Ca 2+ channels on CB 1 receptor inverse agonist/antagonist SR141716A (20 mg/kg, i.p.)-induced vomiting. Different groups of least shrews were given an injection of either the corresponding vehicle or varying doses of one of the following: (1) CaMKII inhibitor KN62 (i.p.); (2) the L-type Ca 2+ channel (LTCC) inhibitors nifedipine (s.c.) and amlodipine (i.p.); (3) store-operated Ca 2+ entry blockers YM 58483 (i.p.) and MRS 1845 (i.p.); and (4) the TRPV1R agonist resiniferatoxin (RTX) (s.c.), 30 min prior to SR141716A (20 mg/kg, i.p.) injection. Emetic parameters were recorded for the next 30 min. The frequency of emesis ( A , C , E , G , I , K ) was analyzed using Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and presented as mean ± SEM. The percentage of shrews vomiting ( B , D , F , H , J , L ) was analyzed using the chi-square test and presented as mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 0 mg/kg. The number of animals in each group is presented at the top of the corresponding column.

Article Snippet: The following drugs were used for the present studies: SR141716A, H-89, U0126, PD98059, AR-A014418, SB216763, LY294002, U73122, GF109203X, KN62, amlodipine, YM-58483, MRS-1845, and RTX were purchased from Tocris (Minneapolis, MN, USA); nifedipine and sulpiride were purchased from Sigma-Aldrich (St. Louis, MO, USA); dantrolene and 2-APB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and palonosetron was kindly provided by Helsinn Health Care (Lugano, Switzerland).

Techniques: Membrane, Injection

Journal: Journal of Extracellular Vesicles

Article Title: Sublytic Activity of a Pore‐Forming Protein From Commensal Bacteria Causes Epigenetic Modulation of Tumour‐Affiliated Protein Expression

doi: 10.1002/jev2.70149

Figure Lengend Snippet: Effect of ClyA + OMVs on CAMK2A and EZH2 protein levels in HCT8 cells. (a) Immunoblot analysis showing the levels of CAMK2A and phosphoEZH2 (threonine‐487) in HCT8 cells after 6 h of treatment with (1) vehicle (20 mM Tris‐HCl, pH 8.0), (2) ClyA + OMVs or (3) ClyA − OMVs. (b, c) Quantification of CAMK2A and pEZH2‐Thr487 protein levels, normalised to β‐actin levels data obtained as shown in (a) from at least two independent experiments, and relative to levels in cells treated with vehicle (20 mM Tris‐HCl). Statistical analysis as described in Section . (d) Effect of ClyA + OMVs on EZH2 protein stability. HCT8 cells were pre‐treated with cycloheximide (50 µg/mL) for 4 h and subsequently treated with ClyA + OMVs (lanes 2–7) or ClyA − OMVs (lanes 9–14). Cell lysates were collected at different time points (0, 2, 4, 6, 8, 10 h) and the levels of EZH2 were assessed by immunoblot analysis using a polyclonal rabbit anti‐EZH2 antibody. (e) Quantification of EZH2 protein levels, normalised against β‐actin, from immunoblot detection experiments as shown in (d). Data compiled from three independent experiments are displayed relative to data from cells treated with vehicle (20 mM Tris‐HCl) and bar graphs show mean ± s.d. Statistical analysis as described in Section . (f) Immunoblot analysis showing EZH2 levels without (lanes 1 and 5) or with (lanes 2–4) pretreatment with the CAMK2A inhibitor KN62 (25 µM) for 2 h, followed by 6 h of treatment with ClyA + OMVs (lane 3) or ClyA − OMVs (lane 4). (g) Quantification of EZH2 protein levels, normalised against β‐actin with bar graphs showing mean ± s.d from two independent experiments. Statistical analysis as described in Section . (h) Levels of EZH2, H3K27me3, Histone H3 and β‐actin in HCT8 cells after treatment with (1) vehicle (20 mM Tris‐HCl, pH 8.0); (2) ClyA + OMVs or (3) ClyA − OMVs for 6 h. Cell lysates were analysed by immunoblotting using specific antibodies for each protein. (I, j) Quantification of EZH2 and H3K27me3 protein levels normalised against β‐actin and Histone H3, respectively. Data from two independent experiments are presented as mean ± s.d. and statistical analysis was done as described in Section . (k) Effect on EZH2 protein levels in colon intestinal organoids treated with (1) vehicle, (2) ClyA + OMVs and (3) ClyA − OMVs during 24 h. (l) Quantified EZH2 protein levels were normalised against β‐actin and are shown as mean ± s.d. of three independent experiments relative to vehicle control set to 1.0. Statistical analysis as described in Section .

Article Snippet: Overnight seeded HCT8, HCT116 and CaCo2 cells (3 × 10 5 ) in a 6‐well plate (Thermo Scientific) were treated with 100 μg/mL of ClyA + OMVs, ClyA − OMVs and ΔlpxM OMVs for 6 h. For the CAMK2A inhibitor experiment, HCT8 cells were pretreated with KN62 (25 μM, Selleckchem USA) for 2 h, followed by treatment with 100 μg/mL of OMVs for 6 h. After treatment, the cells, organoids or animal tumour tissues were lysed in ice‐cold lysis buffer (25 mM Tris‐HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X‐100, 1 mM DTT, 0.5% NP‐40) supplemented with protease (Roche) and phosphatase inhibitor cocktail (Roche).

Techniques: Western Blot, Control

Journal: Redox biology

Article Title: Selective nitration of Hsp90 acts as a metabolic switch promoting tumor cell proliferation.

doi: 10.1016/j.redox.2024.103249

Figure Lengend Snippet: Fig. 5. Nitrated Hsp90 proliferative activity is mediated by activation of P2X7R. (A) Schwannoma (MN-Schwann cells), and (B) normal Schwann cell growth was assessed in the presence and absence of the P2X7R inhibitor KN-62 (10 μM). (C–D) Recombinant proteins were intracellularly delivered to schwannoma and normal Schwann cells and the cells incubated for 24 and 48 h in the presence and absence of the P2X7R inhibitor KN62 (10 μM). The ECAR and glycolytic parameters of (E–G) schwannoma cells, and (H–J) normal Schwann cells were measured 24 h after delivery. Data is shown using Tukey’s representation with the median indicated in the box plot and outliers indicated with dots (n = 3–8 with 8 replicates). *p < 0.05 versus Hsp90 by one-way ANOVA with Dunnett’s multiple com parisons post hoc test.

Article Snippet: The P2X7R inhibitor KN62 (10 μM, Cat. no. S7422, Selleck Chemicals) was dissolved in DMSO and administred at the final concentration indicated.

Techniques: Activity Assay, Activation Assay, Recombinant, Incubation