Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Effects of Aβ 25-35 on System X c − gene expression in U373 cells. Cells were treated with Aβ 25-35 (50 µM) for 4, 8, and 16 h. After incubation at 37 °C, cells were homogenized, and total RNA has been purified to evaluate mRNA content of System X c − by RT-qPCR. Results are computed relative to GAPDH content, taken as a housekeeping gene. Bars are the means ± SEM from three separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to define significant differences. * p ≤ 0.05 vs. CTRL.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Expressing, Incubation, Purification, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Effects of Aβ 25-35 on System X c − protein expression in U373 cells. Cells were treated with Aβ 25-35 (50 µM) for 24 h. The graph shows the densitometric analysis of the western blots for each sample. Data are computed relative to the internal housekeeping gene (actin) and are the means ± SEM from three separate experiments, each carried out in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to define significant differences. ** p ≤ 0.01 vs. CTRL.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Expressing, Western Blot

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: System X c − localization in Aβ 25-35 -treated U373. Cells were treated with Aβ 25-35 (50 µM) for 24 h and subjected to immunofluorescence staining using anti-System X c − 1:100 (OriGene, green) antibodies as reported in Materials and Methods. Nuclei (blue) are stained with Hoechst 33342.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Immunofluorescence, Staining

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Effects of Aβ 25-35 and System X c − activity on release of extracellular glutamate in differentiated SH-SY5Y/U373 co-cultures. Cells were treated for 24 h and the glutamate assay was performed as specified in the Materials and Methods. The graph shows the extracellular release of glutamate (µM). Data are calculated relative to a glutamate standard curve and are the means ± SEM from three separate experiments, each carried out in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to define significant differences. *** p ≤ 0.001 between CTRL and Aβ 25-35 ; ** p < 0.01 between Aβ 25-35 and Aβ 25-35 + SSZ.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Activity Assay, Glutamate Assay

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Role of System X c − and NMDA receptor activation on the viability of Aβ-treated SH-SY5Y differentiated cells. ( A ) SH-SY5Y cells were grown in mono-cultures and treated for 24 h with Aβ 25-35 (50 μM). ( B ) SH-SY5Y cells were co-cultured with U373 cells and treated for 24 h with Aβ 25-35 (50 μM) alone or in the presence of either SSZ (300 μM) or MK801 (10 μM). MTT cell viability assay was carried out as specified in Materials and Methods. The histograms show the percentage of living cells, and the rate of reduction was calculated by setting the control (CTRL) equal to 100%. Values are the means ± SEM from three separate experiments, each performed in duplicate. One-way ANOVA, followed by Bonferroni’s test, was used to determine significant differences. NS not significant; ** p ≤ 0.01 between CTRL and Aβ 25-35 ; ** p ≤ 0.01 between Aβ 25-35 and Aβ 25-35 + SSZ; * p ≤ 0.05 between Aβ 25-35 and Aβ 25-35 + MK801.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Activation Assay, Cell Culture, Viability Assay

Journal: Antioxidants

Article Title: Amyloid-β 25-35 Induces Neurotoxicity through the Up-Regulation of Astrocytic System X c −

doi: 10.3390/antiox10111685

Figure Lengend Snippet: Proposed model for Aβ-dependent neurodegeneration during AD. In astrocytes, Aβ 25-35 elicits an antioxidant response by transcriptionally inducing Nrf2-driven ARE genes, such as CAT, HO-1, SOD1, SOD2, GPX3, GCLC, and System X c − . While the L-cystine import through System X c − is crucial to protection from oxidative stress (e.g., GSH production), the export of glutamate may cause neurodegeneration through the activation of NMDAr on neuronal cells. For more details see text.Abbreviations: Aβ, amyloid-β; ARE, antioxidant responsive element; CAT, catalase; GCLC, glutamate-cysteine ligase; GPX3, glutathione peroxidase; GSH, reduced glutathione; HO-1, heme-oxygenase-1; NMDAr, N-methyl-D-aspartate receptor; Nrf2, nuclear factor erythroid 2-related factor 2; SOD, superoxide dismutase.

Article Snippet: For Western blot analysis and immunofluorescence, the following primary antibodies were used: anti-actin 1:1000 (a2066 Sigma-Aldrich; Milan, Italy), anti-Nrf2 (ab31163 Abcam), anti-Lamin A (ab26300 Abcam; Milan, Italy), anti-System X c − (TA301518 OriGene; Bologna, Italy).

Techniques: Activation Assay

Journal: International Journal of Genomics

Article Title: EIF2S1 Silencing Impedes Neuroblastoma Development Through GPX4 Inactivation and Ferroptosis Induction

doi: 10.1155/2024/6594426

Figure Lengend Snippet: EIF2S1 silencing increases cellular ROS and induces ferroptosis features in neuroblastoma cells. (a) Intracellular-free Fe 2+ levels in SK-N-SH and IMR-32 cells after EIF2S1 silencing. (b) Malondialdehyde (MDA) levels in SK-N-SH and IMR-32 cells after EIF2S1 silencing. (c) Reactive oxygen species (ROS) levels in SK-N-SH and IMR-32 cells after EIF2S1 silencing. (d) Western blot analysis of GPX4 and SLC7A11 protein levels in SK-N-SH and IMR-32 cells after EIF2S1 silencing. sh-EIF2S1 versus sh-NC ; ⁣ ∗∗∗ indicates p < 0.0001.

Article Snippet: These membranes were subsequently placed in an ice-cold buffer containing primary antibodies and left to incubate overnight, including SLC7A11 (Cat#K009230P, Solarbio Life Sciences, Beijing, China), EIF2S1 (Cat# K010094P, Solarbio), GPX4 (Cat#K003083P, Solarbio), N-Myc (Cat#ab24193, Abcam), and antitubulin (Cat#K200059M, Solarbio).

Techniques: Western Blot