Furthermore, CBR-470-1-induced mRNA and proteins appearance of Nrf2 pathway genes, and mRNA in stable SH-SY5Y neuronal cells with Nrf2 shRNA (sh-Nrf2) or a lenti-CRISPR/Cas9-Nrf2 KO construct (ko-Nrf2), as well as in the parental control cells (Pare), was shown (A); Cells were treated with CBR-470-1 (CBR, 10 M) or the vehicle control (Veh) for applied time periods, expression of outlined mRNAs and proteins was shown (BCD); Alternatively, cells were pre-treated for 2h with CBR-470-1 (CBR, 10 M) or the vehicle control (Veh), followed by MPP+ (3 mM) activation for 48h, cell viability and death were tested by CCK-8 (E) and medium LDH release (F) assays, respectively. guarded SH-SY5Y cells from MPP+. In Keap1 or PGK1 knockout SH-SY5Y cells,CBR-470-1 failed to offer further cytoprotection against MPP+. Collectively PGK1 inhibition by CBR-470-1 protects SH-SY5Y cells from MPP+ via activation of the Keap1-Nrf2 cascade. and (Physique 1F). The mRNA levels were, however, unchanged after CBR-470-1 treatment in SH-SY5Y cells (Physique 1F). Increased protein expression of HMOX1, NQO1 and SOD1 was also detected inCBR-470-1-treated cells (Physique 1G). CBR-470-1 inhibits MPP+-induced oxidative injury in SH-SY5Y neuronal cells In line with previous studies [2, 21C23], we found that MPP+ induced oxidative injury in SH-SY5Y neuronal cells, causing strong lipid peroxidation (TBAR activity increase, Physique 2A), single strand DNA (ssDNA) accumulation (Physique 2B) and mitochondrial depolarization (JC-1 green fluorescence intensity increase, Physique 2C). Importantly, pretreatment with CBR-470-1(10 M, 2h) in SH-SY5Y cells attenuated MPP+-induced oxidative injury (Physique 2AC2C). Open in a separate window Physique 2 CBR-470-1 inhibits MPP+-induced oxidative injury in SH-SY5Y neuronal cells. SH-SY5Y neuronal cells were pre-treated for 2h with CBR-470-1 (CBR, 10 M) or the vehicle control (Veh), followed by MPP+ (3 mM) activation, cells were further cultured for applied time periods, relative lipid peroxidation levels (A), single strand DNA contents (B) and mitochondrial depolarization(JC-1 green fluorescence intensity, (C) were tested, and then cell viability and death examined by CCK-8 (D) and medium LDH release (E) assays, respectively. Cell apoptosis was evaluated by the assays pointed out in the text (FCH).Veh stands for the vehicle control. Mock Goserelin stands for MPP+ single treatment (no pretreatment).Ctrl stands for no MPP+ activation. Bars stand for mean standard deviation (SD, n=5). * mRNA (sh-Nrf2 cells, Physique 3A). Furthermore, a lenti-CRISPR/Cas9-Nrf2 KO construct was utilized to knockout (KO) Nrf2 in SH-SY5Y cells (ko-Nrf2 cells, Physique 3A). As shown, CBR-470-1-induced cytosolic accumulation of Nrf2 protein was completely blocked in sh-Nrf2 cells and ko-Nrf2 cells (Physique 3B). Furthermore, CBR-470-1-induced mRNA and protein expression of Nrf2 pathway genes, and mRNA in stable SH-SY5Y neuronal cells with Nrf2 shRNA (sh-Nrf2) or a lenti-CRISPR/Cas9-Nrf2 KO construct (ko-Nrf2), as well as in the parental control cells (Pare), was shown (A); Cells were treated with CBR-470-1 (CBR, 10 M) or the vehicle control (Veh) for applied time periods, expression of outlined mRNAs and proteins was shown (BCD); Alternatively, cells were pre-treated for 2h with CBR-470-1 (CBR, 10 M) or the vehicle control (Veh), followed by MPP+ (3 mM) activation for 48h, cell viability and death were tested by CCK-8 (E) and Goserelin medium LDH release (F) assays, respectively. Expression of listed proteins was quantified and normalized to the loading control (B, D). Bars stand for mean standard deviation (SD, n=5). * mRNA (Physique 4A) and protein (Physique 4A) decreased by over 95% in both sh-PGK1 cells and ko-PGK1 cells. Nrf2 protein accumulated with PGK1 silencing or KO (Physique 4B), leading to increased ARE luciferase activity (Physique 4C) and expression of Nrf2 pathway genes (and mRNA (Physique 5A) and protein (Physique 5B). Keap1 KO resulted in Nrf2 protein stabilization and accumulation (Physique 5B), increased ARE activity (Physique 5C), and expression of Nrf2 pathway genes (and and total LDH). Cell viability The differentiated SH-SY5Y cells were cultured onto six well-tissue plates (at 1105 cells per well). Following the applied MPP+ treatment, cell viability was quantified via a cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan), and its optical density (OD) values Goserelin tested at 550 nm. Western blotting and co-immunoprecipitation (co-IP) The detailed protocols of Western blotting were previously reported [30, 31]. In brief, lysate proteins were separated by SDS-PAGE gels [32], transferred to PVDF blots(Millipore, Shanghai, China). The blots were blocked and incubated with the designated main and secondary antibodies. An ECL reagent kit (Pierce, Shanghai, China) was applied to detect the protein band under X-ray films. Data quantification was carried out by an ImageJ software (NIH). For the co-IP studies, the quantified protein lysates (1, 000 g for each treatment) were pre-cleared and incubated with anti-Keap1 antibody [33]. Keap1-Nrf2 complex GRF2 was captured by the G-Sepharose (Beads, Sigma), tested by Western blotting. Screening nuclear portion lysates was explained in our previous studies [30, 31] Caspase-3 activity SH-SY5Y cells were cultured onto six well-tissue plates (at 1105 cells per well). Following the applied MPP+ treatment, the caspase-3 activity was tested Goserelin by a commercial fluorometric caspase-3 assay kit (Beyotime Biotechnology, Wuxi, China) [34], using the previously-described protocol [31]. Cell apoptosis analyses.