camkii inhibitor myristoylated autocamtide  (Tocris)

Journal: The Journal of Biological Chemistry

Article Title: A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations

doi: 10.1016/j.jbc.2026.111365

Figure Lengend Snippet: Serine/threonine protein kinases regulating PEAK3 S69 phosphorylation. A , consensus motifs of serine/threonine protein kinases with selectivity corresponding to the PEAK3 S69 region. Phosphorylation sites (position 0) are highlighted in pink and key features highlighted are arginine or lysine at position −3 ( orange ) and hydrophobic residues (ϕ) at +1 and −5 position ( blue ). X = any amino acid. Motifs sourced from Johnson et al. and PhosphoSitePlus ( , ). B , role of PKCs. MCF-10A/PEAK3 cells were treated with Go6850 or Go6976 and cell lysates subjected to Western blotting as indicated. Results are representative of four independent experiments. Histogram indicates the relative level of PEAK3 p-S69 normalized for HA and expressed relative to the DMSO control, which was arbitrarily set to 1.0. Data represent mean ± S.E.M., with 'au' indicating arbitrary units. ∗ p < 0.05, ∗∗ p < 0.01, by ratio paired t test. C , role of CaMKII as determined by pharmacological inhibition. MCF-10A/PEAK3 cells were treated with RA306 or vehicle control and cell lysates analyzed by Western blotting as indicated. Data are representative of three independent experiments. Results in C are derived from the same experiment and use the same DMSO control. Histogram indicates the relative level of PEAK3 p-S69 as mean ± S.E.M., ∗∗ p < 0.01, by ratio paired t test. D , role of CaMKII as determined by knockdown. CaMKIID and G were subject to siRNA-mediated knockdown (KD) in MCF-10A/PEAK3 cells and PEAK3 p-S69 assayed by Western blotting as indicated. Data are representative of three independent experiments. Histogram indicates the relative level of PEAK3 p-S69 as mean ± S.E.M. over three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, by ratio paired t test.

Article Snippet: The following reagents/inhibitors were used in this study: lambda protein phosphatase (New England Biolabs, P0753S), animal-free recombinant human EGF (PeproTech, AF-100–15), insulin from bovine pancreas (Merck, I-1882), CaMKII inhibitor RA306 (custom synthesized by Reagency, RGNCY-0117, 1 μM final), PKA Inhibitor 14 to 22 Amide, Cell-Permeable, Myristoylated (Calbiochem, 476,485, 10 μM final), DMSO (Sigma, d8418, 0.1% final), and MedChemExpress-sourced Akt inhibitor MK-2206 dihydrochloride (HY-10358, 100 nM final) and trametinib (HY-10999, 10 nM final).

Techniques: Phospho-proteomics, Western Blot, Control, Inhibition, Derivative Assay, Knockdown

Journal: Advanced Science

Article Title: Mechanical Stress Induced NOX2 Promotes Endothelial Dysfunction in Ventilator‐Induced Lung Injury: Potential Treatment with Quercetin

doi: 10.1002/advs.202502639

Figure Lengend Snippet: High cyclic stretch disrupts endothelial junctions via NOX2/ROS/CaMKII/ERK1/2 Axis. A,B) Representative DHE fluorescent images (A) and quantitative analysis (B) in control or H 2 O 2 treated HAECs co‐incubated with or without KN93 (a selective inhibitor for CaMKII). Scale bars: 30 µm. n = 6. C,D) Representative Western blot (C) and the quantitative analysis (D) of phosphorylations of CaMKII and ERK1/2 in control or H 2 O 2 treated HAECs co‐incubated with or without KN93. Data are presented as the fold changes relative to the Control group. n = 4. E–G) Representative immunofluorescent images of VE‐cadherin (E) and ZO‐1 (F) and the quantitative analysis (G) of ZO‐1 and VE‐cadherin in control or H 2 O 2 treated HAECs co‐incubated with or without KN93. Nuclei are counterstained with DAPI. Scale bars: 20 µm. The fluorescence intensity of ZO‐1 and VE‐cadherin is quantified using Image J software. n = 5–6. H,I) Representative Western blot (H) and the quantitative analysis (I) of phosphorylations of CaMKII and ERK1/2 in HAECs pre‐treated with or without KN93 under static or CS conditions. Data are presented as the fold changes relative to the static group. n = 4. J,K) Representative Western blot (J) and the quantitative analysis (K) of phosphorylations of CaMKII and ERK1/2 in HAECs transfected with siNC or siNOX2 under static and CS conditions. Data are presented as the fold changes relative to the ST + siNC group. n = 4. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 for the indicated comparisons. H 2 O 2 : hydrogen peroxide; P‐CaMKII: phosphorylated CaMKII; P‐ERK1/2: phosphorylated ERK1/2; VE‐cad: VE‐cadherin; DAPI: 4′,6‐diamidino‐2‐phenylindole; ST: static; CS: cyclic stretch; NC: negative control.

Article Snippet: To explore the effects of ROS on cell function, HAECs were treated with different concentrations of hydrogen peroxide (H 2 O 2 ) (0, 100 μM, 200 μM, 500 μM) [ , , ] for 24 h before protein collection; For CaMKII inhibition, HAECs were co‐incubated with H 2 O 2 (500 μM) and the CaMKII inhibitor KN93 [ , ] (1 μM, Cat#: 139298‐40‐1, MCE) for 24 h; For ROS scavenging, the antioxidant drug quercetin (1 μM) was co‐incubated with H 2 O 2 hydrogen peroxide (500 μM) for 24 h. For experiments related to CS loading, HAECs were pre‐treated with KN93 (1 μM) or quercetin (1 μM) and then subjected to mechanical loading.

Techniques: Control, Incubation, Western Blot, Fluorescence, Software, Transfection, Negative Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mechanical Stress Induced NOX2 Promotes Endothelial Dysfunction in Ventilator-Induced Lung Injury: Potential Treatment with Quercetin.

doi: 10.1002/advs.202502639

Figure Lengend Snippet: Figure 3. Elevated ROS induce CaMKII/ERK1/2 signaling pathway activation in mechanical ventilation-induced lung injury (VILI). A) Venn diagram of shared genes in two sets (GSE226807 and GSE114132) of differentially expressed genes in lungs from sham control and MV. B,C) Bubble plot of Gene Ontology (GO) enrichment analysis for 884 genes differentially expressed in both sets. The x-axis represents the Rich Factor, indicating the degree of enrichment. The color scale indicates the -log2 (FDR), reflecting the significance of enrichment. Biological process (B) and Cellular component (C). D) Bubble plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for 884 genes differentially expressed in both sets. The x-axis represents the Rich Factor, indicating the degree of enrichment. The color scale indicates the -log2 (Q-value), reflecting the significance of enrichment. The differentially expressed genes between H-MV and control groups E). PCA showed the distribution of the samples in the control group and the H-MV group F). The downregulated and unregulated genes in H-MV group compared to control group G). The volcano plot visually displays the distribution of differential genes H). Gene Ontology (GO) analysis between H-MV group and control group I). KEGG enrichment analysis between H-MV group and control group J). K) IPA predicts the signaling regulatory network associated with endothelial function, in which the NOX2/ROS/CaMKII/ERK1/2 signaling axis is highlighted in red. L,M) Representative Western blot (L) and the quantitative analysis (M) of phosphorylations and total expressions of CaMKII and ERK1/2 in lung tissue homogenates from the Control, L-MV, and H-MV groups respectively. Data are presented as the fold changes relative to the Control group. n = 6. N) Correlation analysis of p-CaMKII expression and DHE mean intensity defined ROS levels indicating that ROS level is positive correlated with p-CaMKII expression. R = 0.5973, p = 0.0187. n = 15. O) Correlation analysis of p- ERK1/2 expression and DHE mean intensity defined ROS levels indicating that ROS level is positive correlated with ERK1/2 expression. R = 0.7000, p = 0.0048. n = 15. P,Q) Representative Western blot (P) and the quantitative analysis (Q) of phosphorylations of CaMKII and ERK1/2 in HAECs stimulated with H2O2 at concentrations of 0 μM, 100 μM, 200 μM, and 500 μM respectively. Data are presented as the fold changes relative to the 0 nM group. n = 3. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. L-MV: low mechanical ventilation; H-MV: high mechanical ventilation; P-CaMKII: phosphorylated CaMKII; P-ERK1/2: phosphorylated ERK1/2; H2O2: hydrogen peroxide.

Article Snippet: To explore the effects of ROS on cell function, HAECs were treated with different concentrations of hydrogen peroxide (H2O2) (0, 100 μM, 200 μM, 500 μM)[67–69] for 24 h before protein collection; For CaMKII inhibition, HAECs were co-incubated with H2O2 (500 μM) and the CaMKII inhibitor KN93[70,71] (1 μM, Cat#: 139298-40-1, MCE) for 24 h; For ROS scavenging, the antioxidant drug quercetin (1 μM)was co-incubated withH2O2hydrogen peroxide (500 μM) for 24 h. For experiments related to CS loading, HAECs were pre-treated with KN93 (1 μM) or quercetin (1 μM) and then subjected to mechanical loading.

Techniques: Activation Assay, Control, Western Blot, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mechanical Stress Induced NOX2 Promotes Endothelial Dysfunction in Ventilator-Induced Lung Injury: Potential Treatment with Quercetin.

doi: 10.1002/advs.202502639

Figure Lengend Snippet: Figure 4. High cyclic stretch disrupts endothelial junctions via NOX2/ROS/CaMKII/ERK1/2 Axis. A,B) Representative DHE fluorescent images (A) and quantitative analysis (B) in control or H2O2 treated HAECs co-incubated with or without KN93 (a selective inhibitor for CaMKII). Scale bars: 30 μm. n = 6. C,D) Representative Western blot (C) and the quantitative analysis (D) of phosphorylations of CaMKII and ERK1/2 in control or H2O2 treated HAECs co-incubated with or without KN93. Data are presented as the fold changes relative to the Control group. n = 4. E–G) Representative immunofluorescent images of VE-cadherin (E) and ZO-1 (F) and the quantitative analysis (G) of ZO-1 and VE-cadherin in control or H2O2 treated HAECs co-incubated with or without KN93. Nuclei are counterstained with DAPI. Scale bars: 20 μm. The fluorescence intensity of ZO-1 and VE-cadherin is quantified using Image J software. n = 5–6. H,I) Representative Western blot (H) and the quantitative analysis (I) of phosphorylations of CaMKII and ERK1/2 in HAECs pre- treated with or without KN93 under static or CS conditions. Data are presented as the fold changes relative to the static group. n = 4. J,K) Representative Western blot (J) and the quantitative analysis (K) of phosphorylations of CaMKII and ERK1/2 in HAECs transfected with siNC or siNOX2 under static and CS conditions. Data are presented as the fold changes relative to the ST + siNC group. n = 4. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. H2O2: hydrogen peroxide; P-CaMKII: phosphorylated CaMKII; P-ERK1/2: phosphorylated ERK1/2; VE-cad: VE-cadherin; DAPI: 4′,6-diamidino-2-phenylindole; ST: static; CS: cyclic stretch; NC: negative control.

Article Snippet: To explore the effects of ROS on cell function, HAECs were treated with different concentrations of hydrogen peroxide (H2O2) (0, 100 μM, 200 μM, 500 μM)[67–69] for 24 h before protein collection; For CaMKII inhibition, HAECs were co-incubated with H2O2 (500 μM) and the CaMKII inhibitor KN93[70,71] (1 μM, Cat#: 139298-40-1, MCE) for 24 h; For ROS scavenging, the antioxidant drug quercetin (1 μM)was co-incubated withH2O2hydrogen peroxide (500 μM) for 24 h. For experiments related to CS loading, HAECs were pre-treated with KN93 (1 μM) or quercetin (1 μM) and then subjected to mechanical loading.

Techniques: Control, Incubation, Western Blot, Software, Transfection, Negative Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Mechanical Stress Induced NOX2 Promotes Endothelial Dysfunction in Ventilator-Induced Lung Injury: Potential Treatment with Quercetin.

doi: 10.1002/advs.202502639

Figure Lengend Snippet: Figure 5. Quercetin effectively prevents mechanical stretch-induced endothelial dysfunction by scavenging ROS in vitro. A–C) Representative immunoflu- orescent images of ZO-1 (A) and VE-cadherin (B) and the quantitative analysis (C) in control or H2O2 treated HAECs co-incubated with or without quercetin. Nuclei are counterstained with DAPI. Scale bars: 20 μm. The fluorescence intensity of ZO-1 and VE-cadherin is quantified using Image J software. n = 6. Representative Western blot and the quantitative analysis of NOX2 (D). n = 4. E,F) Representative Western blot (E) and the quantitative analysis (F) of phosphorylations of CaMKII and ERK1/2 in HAECs pre-treated with or without quercetin under static or CS. Data are presented as the fold changes relative to the static group. n = 4. G,H) Representative Western blot (G) and the quantitative analysis (H) of protein expressions of ZO-1 and VE-cadherin in HAECs pre-treated with or without quercetin under static or CS. Data are presented as the fold changes relative to the static group. n = 4. I–K) Representative immunofluorescent images of ZO-1 (I) and VE-cadherin (J) and the quantitative analysis (K) in HAECs pre-incubated with or without quercetin under static or CS. Nuclei are counterstained with DAPI. Scale bars: 20 μm. The fluorescence intensity of ZO-1 and VE-cadherin is quantified using Image J software. n = 5-6. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons. L-MV: low mechanical ventilation; H-MV: high mechanical ventilation; P-CaMKII: phosphorylated CaMKII; P-ERK1/2: phosphorylated ERK1/2; VE-cad: VE-cadherin; H2O2: hydrogen peroxide; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: To explore the effects of ROS on cell function, HAECs were treated with different concentrations of hydrogen peroxide (H2O2) (0, 100 μM, 200 μM, 500 μM)[67–69] for 24 h before protein collection; For CaMKII inhibition, HAECs were co-incubated with H2O2 (500 μM) and the CaMKII inhibitor KN93[70,71] (1 μM, Cat#: 139298-40-1, MCE) for 24 h; For ROS scavenging, the antioxidant drug quercetin (1 μM)was co-incubated withH2O2hydrogen peroxide (500 μM) for 24 h. For experiments related to CS loading, HAECs were pre-treated with KN93 (1 μM) or quercetin (1 μM) and then subjected to mechanical loading.

Techniques: In Vitro, Control, Incubation, Software, Western Blot