Journal: Redox Biology

Article Title: Brain ischemic preconditioning protects against ischemic injury and preserves the blood-brain barrier via oxidative signaling and Nrf2 activation

doi: 10.1016/j.redox.2018.05.001

Figure Lengend Snippet: Diagram showing effects of 4-HNE on Keap1- and GSK3β-mediated Nrf2 degradation. (A) Diagram showing the effects of 4-HNE on the “conforming cycle” in Keap1-mediated Nrf2 degradation. Left panel: under normal conditions, Keap1 binds to Nrf2 and guides it to the proteasome. At the proteasome, Keap1 is disassociated from Nrf2 and is recycled to bind newly synthesized Nrf2. Right panel: we discovered that 4-HNE interrupts both Keap1-Nrf2 binding and their disassociation, leading to a decrease in both Nrf2 degradation and Keap1 recycling; the latter then results in accumulation of newly synthesized Nrf2 which binds EpRE. We also showed that the inhibitory effect of 4-HNE on Keap1-Nrf2 binding is dependent on C288. (B) Diagram showing the effects of 4-HNE on GSK3β-mediated Nrf2 degradation via both direct and indirect pathways. In the indirect pathway, both 4-HNE and 4-HHE decrease PTEN activity, which further induces Akt phosphorylation and activation. Activated p-Akt inactivates GSK3β by phosphorylating S9 of GSK3β. We also discovered the direct pathway, in which 4-HNE directly inactivates GSK3β by adducting the C199 residue.

Article Snippet: For each reaction, 400 ng recombinant PTEN (rPTEN, R&D, Minneapolis, MN) was incubated with 4-HNE or 4-HHE in 50 mM tricine and 100 mM NaCl (pH = 8.0) assay buffer for 30 min at room temperature.

Techniques: Synthesized, Binding Assay, Activity Assay, Activation Assay

Journal: Scientific Reports

Article Title: A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition

doi: 10.1038/s41598-021-83857-y

Figure Lengend Snippet: GSK-J4 induces the expression of ATF4 protein and its downstream target genes. ( A ) Analysis of GSK-J4-regulated gene expression by reverse transcription-quantitative PCR (RT-qPCR). Cells (786-O, PRETEC and HCT116) were treated with the indicated concentrations (0, 5 or 30 μM) of GSK-J4 for 20 h and their isolated total RNA was analyzed. Bars show the mean of biological triplicates (N = 3) with standard error of the mean (S.E.M.) bars and p-value asterisks (*< 0.05; **< 0.01; ***< 0.001). Asterisks are placed vertically. Fold changes were calculated against the DMSO control of each cell type at 1.0 after normalizing against the PPIA housekeeping gene. Primer sequences are presented in Table . ( B ) Western blot analysis. Total cell extracts from HCT116 treated with GSK-J4 or tunicamycin (Tm) for 20 h at the indicated concentrations were analyzed with the antibodies denoted on the right. Arrows indicate specific bands. Vertical short bar indicates the phosphorylated (sifted up) PERK band. Bands were quantified and relative values to the control (DMSO) are presented. Both short and long exposure photos are presented for LC3B. ( C ) Similarly treated HCT116 cells were also analyzed by immunofluorescence (IF) staining with antibodies against ATF4 or CHOP. Fluorescence images after staining with Alexa 488-conjugated secondary antibody and DAPI ( D ) are shown. Staining was performed under the same conditions. Images were captured by the exposure of 1000 ms for immunostaining and 80 ms for DAPI.

Article Snippet: Primary renal proximal tubule epithelial cells (PRETEC) (ATCC, PCS-400-010) were cultured in the recommended media (ATCC, PCS-400-030 and PCS-400-040) and used within tenfold expansion in cell number.

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Control, Western Blot, Immunofluorescence, Staining, Fluorescence, Immunostaining