Journal: Molecular medicine reports

Article Title: Theaflavin attenuates cerebral ischemia/reperfusion injury by abolishing miRNA‑128‑3p‑mediated Nrf2 inhibition and reducing oxidative stress.

doi: 10.3892/mmr.2019.10755

Figure Lengend Snippet: Figure 4. Downregulation of miRNA‑128‑3p and upregulation of Nrf2 promote cell proliferation and inhibit apoptosis and oxidative stress. (A) Relative expression level of Nrf2 after upregulation of Nrf2 (OE‑Nrf2). (B) Representative neurosphere images in NSCs subjected to OGD/R after downregulation or upregulation of miRNA‑128‑3p (with inhibitor and mimics, respectively), or upregulation of Nrf2 (OE‑Nrf2). (C) Quantitative analysis of neurosphere diameter by Image J software. (D) Cell Counting Kit‑8 (CCK‑8) assay shows the cell viability of NSCs subjected to OGD/R after downregulation or upregulation of miRNA‑128‑3p (with inhibitor and mimics, respectively), or upregulation of Nrf2 (OE‑Nrf2). (E) Flow cytometry shows the apoptosis of NSCs subjected to OGD/R after downregulation of miRNA‑128‑3p (Inhibitor) or upregulation of Nrf2 (OE‑Nrf2). (F) Quantitative analysis of the apoptotic rate. (G) ROS levels in NSCs subjected to OGD/R after after downregulation or upregulation of miRNA‑128‑3p (with inhibitor and mimics, respectively), or upregulation of Nrf2 (OE‑Nrf2). (H) MDA content in NSCs subjected to OGD/R after downregulation of miRNA‑128‑3p (Inhibitor) or upregulation of Nrf2 (OE‑Nrf2). (I) GSH‑Px levels in NSCs subjected to OGD/R after downregulation or upregulation of miRNA‑128‑3p (with inhibitor and mimics, respectively), or upregulation of Nrf2 (OE‑Nrf2). (J) SOD activity in NSCs subjected to OGD/R after downregulation of miRNA‑128‑3p (Inhibitor) or upregulation of Nrf2 (OE‑Nrf2). All assays were performed in triplicate. Data are expressed as the mean ± SD. *P<0.05, **P<0.01. Nrf2, nuclear factor (erythroid‑derived 2)‑related factor 2; NSCs, neural stem cells; OGD/R, oxygen‑glucose deprivation and reoxygenation; MDA, malondialdehyde; GSH‑Px, glutathione peroxidase; SOD, superoxide dismutase.

Article Snippet: The levels of malondialdehyde (MDA), glutathione peroxidase (GSH‐Px), and superoxide dismutase (SOD) in the rat brain tissues and NSCs were measured using standard assay kits, including the Total SOD Activity Detection kit (Solarbio, Beijing, China), Malondialdehyde (MDA) Content Detection kit (Solarbio), and Glutathione Peroxidase (GSH‐Px) Detection kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.

Techniques: Expressing, Software, CCK-8 Assay, Flow Cytometry, Activity Assay

Journal: Scientific Reports

Article Title: Functional kinomics establishes a critical node of volume-sensitive cation-Cl − cotransporter regulation in the mammalian brain

doi: 10.1038/srep35986

Figure Lengend Snippet: ( A ) Characterization of HEK293 cells with doxycycline (dox)-inducible MYC-KCC3 expression used in the RNAi screen. KCC3 wild type (WT) and KCC3 Thr 991 Ala/Thr 1048 Ala protein expression was induced by 0.1 μg/ml doxycycline in the culture medium for 24 hours . Cell lysates were subjected to Western immunoblot (IB) analysis with the indicated antibodies ( B ) Characterization of anti-KCC3 P-Thr 991 and anti-KCC3 P-Thr 1048 phospho-specific antibodies. 36 hours post-transfection with the indicated FLAG-tagged constructs, HEK293 cells were treated for 30 min with either isotonic conditions or hypotonic high K + conditions. Total cell extracts were subjected to IB analysis with the indicated antibodies. Mutation of these residues to alanine (Ala 991 and Ala 1048 ) prevented phosphorylation and eliminated the phospho-specific antibody signal at both sites. ( C ) Scheme of the RNAi screen using the human Dharmacon SMARTpool siRNA kinome library to identify essential kinase regulators of KCC3 Thr 991 phosphorylation. ( D ) Example of results from the primary siRNA screen. Band density of KCC3 P-Thr 991 from Western blots was quantitated by ImageJ software, and these values were used to calculate the magnitude of KCC3 P-Thr 991 increase or decrease by comparing to values derived from Firefly (FF) luciferase negative controls. The heat map depicts the average scores for each kinase siRNA pool in the screen that decreased (green) or increased (red) the signal of KCC3 P-Thr 991 relative to that of the FF siRNA. See Methods for further details. ( E ) Scattered and sorted robust z-scores of kinase hits from the siRNA primary screen. Several siRNA pools led to a significant decrease in the KCC3 P-Thr 991 signal (>50%, p < 0.01 compared to FF siRNA negative control). ( F ) Summary of kinase hits from the secondary siRNA screen. siRNAs targeting primary screen hits were analyzed for their ability to decrease KCC3 P-Thr 991 without affecting total KCC3 level. The (KCC3 P-Thr 991 )/(total MYC-KCC3) ratio was calculated for each target based on the quantification of immuno-reactive signals in triplicate Western blots, with a value of 100% for FF. Ratios were compared by one-way ANOVA (n = 3, mean ± SEM), with p < 0.01 considered statistically significant.

Article Snippet: To identify genes required for KCC3 P-Thr 991 phosphorylation, a high-throughput RNAi screen was performed in 24-well plates with the human Dharmacon SMARTpool siRNA kinome library targeting 541 kinases and kinase-related genes in which each mRNA is targeted by a pool of siRNAs consisting of a combination of four siRNA duplexes directed at different regions of the gene.

Techniques: Expressing, Western Blot, Transfection, Construct, Mutagenesis, Phospho-proteomics, Software, Derivative Assay, Luciferase, Negative Control