Supplementary MaterialsMultimedia component 3 mmc3. alterations in glucose metabolism and neuronal Sulfosuccinimidyl oleate death. To date, the endogenous molecules that act as intrinsic regulators of neuronal necroptosis under conditions of oxidative stress Sulfosuccinimidyl oleate are unknown. Here, we show that treatment with AA regulates the expression of pro- and antiapoptotic genes. Vitamin C also regulates the expression of RIPK1/MLKL, whereas the oxidation of AA in neurons induces morphological alterations consistent with necroptosis and MLKL activation. The activation of necroptosis by AA oxidation in neurons results in bubble formation, loss of membrane integrity, and ultimately, cellular explosion. These data suggest that necroptosis is a target for cell death induced by vitamin C. and N2acells were generated by CRISPR/Cas9 using CAG-Cas9-2a-RFP and Cas9-ElecD plasmids (Atum, #pD1321-AP) and transfection with Lipofectamine 3000 (Life Technologies). The gRNA target sequences for the murine initiation codons of MLKL and SVCT2 were GCACACGGTTTCCTAGACGC and TGTAGATCATATCCGACCTC, respectively. The cells were selected at 48?h posttransfection using a BD FACSAria III cell sorter. Single-cell RFP was sorted in 96-well plates. MLKL- and SVCT2-deleted colonies were verified by Western blotting. N2a-hSVCT2wt-EYFP, N2a-EGFP, HN33.11-hSVCT2wt-EYFP, and HN33.11-EGFP cells were generated by infection with lentiviral particles as previously described [2]. Stable EYFP- and EGFP-expressing cells were selected at 72?h postinfection by FACS. 2.3. Live-cell microscopy N2a and HN33.11?cells were seeded in 18-mm cover glasses in 12-well plates for 48?h. After treatment with H2O2, the cover was removed, and the plates were placed in a live-cell perfusion chamber. Then, the cells were loaded with fluorescent probes for 10?min and washed with PBS. Finally, the cells were incubated in complete medium and imaged at 37?C and 5% CO2 in a confocal spectral Zeiss LSM 780 live-cell system. The images were acquired in 4D (x: 1024, y: 1024, z: 6 or 10, time, channels: 5, 8-bit) with an objective Plan-Apochromat 63x/1.40 Oil DIC M27. The following fluorescent probes were used: Hoechst Sulfosuccinimidyl oleate 33342 (0.1?g/mL, ex/em (nm) 350/461), Alexa Fluor 488 phalloidin (20?nM, ex/em (nm) 495/518), MitoTracker Red SFN CMXRos (25?nM, ex/em (nm) 579/599), and cellmask (0.3X ex/em (nm) 650/655). Finally, the images were reconstructed in a movie using the Zen lite software (Zeiss). 2.4. Immunocytochemistry and image processing Cells were seeded on coverslips. After treatment, the cells were fixed with 4% paraformaldehyde for 30?min?at room temperature, washed with Tris-phosphate buffer Sulfosuccinimidyl oleate [35] and incubated overnight at room temperature with the following antibodies: anti-SVCT2 (1:50), anti-GLUT1 (1:400), anti-RIPK1 (1:400), anti-RIPK3 (1:50), anti-MLKL (1:400), anti-phospho RIPK1 (1:100) and anti-phospho MLKL (1:100). The cells were incubated at room temperature for 2?h with Cy3 AffiniPure Donkey Anti-Goat IgG, Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG, Cy2 AffiniPure Donkey Anti-Mouse IgG, Cy5 AffiniPure Donkey Anti-Rabbit IgG, Alexa Fluor 488 AffiniPure Donkey Anti-Rat IgG or Cy3 AffiniPure Donkey Anti-Rat IgG (1:200). Hoechst 33342 (1:1000) was used for nuclear staining. The images were acquired using an LSM 780 spectral confocal microscope (Zeiss) or ELIRA S.1 Superresolution Structured Illumination Microscopy (Zeiss). The images were exported in .czi format and processed in Imaris v 9.1 software (Bitplane Inc) for 3D reconstruction, colocalization, morphology and bounding box analysis. The intensity profile was determined with ImageJ software. 2.5. Cell viability assay N2a and HN33.11?cells were supplemented with 200?M AA for 36?h. Then, intracellular oxidation of AA was induced by incubation with 500?M H2O2 for 30?min (or the concentration indicated in the figure). After this time, H2O2 Sulfosuccinimidyl oleate was removed, and the cells were washed with PBS and incubated in complete medium for 3?h. Finally, cell viability was measured by XTT (Biological Industries #20-300-1000) colorimetric analysis. Cell death by loss of plasma membrane integrity was measured by flow cytometry (BD FACSAria III) with 500?nM TOPRO-3 (10?min) [36]. The flow cytometry data were processed with FlowJo software (Tree Star). Nec-1, Nec-1s and zVAD.FMK were used during and after treatment with H2O2. 2.6. Measurement of ROS The cells were trypsinized, resuspended in serum-free DMEM-F12 (GIBCO), incubated for 30?min with 500?nM CellROX Deep Red (Life Technologies) and analyzed by flow cytometry (BD FACSAria III). The flow cytometry data were processed with FlowJo software (Tree Star). 2.7. Intracellular measurement of AA The cells were washed with PBS, trypsinized and resuspended in cold PBS. AA was measured using the ferric reducing (antioxidant) activity and ascorbic acid concentration (FRASC) colorimetric assay (bioassay system #EASC-100) according to the manufacturer’s instructions. 2.8. Western blot analysis N2a and HN33.11?cells were lysed with NP-40 buffer supplemented with a protease/phosphatase.