Journal: Acta Biochimica et Biophysica Sinica

Article Title: Biochemical mechanism of erastin-induced ferroptotic cell death in neuronal cells

doi: 10.3724/abbs.2023058

Figure Lengend Snippet: PDI knockdown protects HT22 cells against erastin-induced ferroptosis (A,B) Levels of dimer and monomer nNOS after treatment with 1 μM erastin for the indicated time intervals (A) or at the indicated concentrations for 8 h (B). (C) Levels of dimer and monomer nNOS after incubation of cell lysates with PDI (1 mg/mL) in vitro at 4°C for 60 min. (D) Effectiveness of PDI-siRNA in reducing cellular PDI protein levels. Cells were transfected with PDI-siRNA for 48 h, and the PDI levels were determined by western blot analysis. (E) Effect of PDI knockdown on erastin-induced nNOS dimerization. Cells were transfected with PDI-siRNA for 24 h prior to treatment with 1 μM erastin for an additional 24 h. nNOS dimer formation was detected by western blot analysis. (F‒J) Effect of PDI knockdown on erastin-induced NO and ROS accumulation. Cells were transfected with PDI-siRNA for 24 h prior to erastin (1 μM) treatment. The levels of NO and ROS after 8 h of erastin treatment were assessed by fluorescence microscopy (F,H), and their quantitative intensity values are shown in G and I. Cell viability changes were analysed after 24 h of erastin treatment (J). Scale bar: 45 μm. Data are presented as the mean±SE. n=3. * P<0.05, ** P<0.01 vs the control group.

Article Snippet: Mouse anti-nNOS and rabbit anti-PDI antibodies were obtained from Solarbio (Beijing, China).

Techniques: Knockdown, Incubation, In Vitro, Transfection, Western Blot, Fluorescence, Microscopy, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Biochemical mechanism of erastin-induced ferroptotic cell death in neuronal cells

doi: 10.3724/abbs.2023058

Figure Lengend Snippet: Cystamine (a PDI inhibitor) protects HT22 cells against erastin-induced ferroptosis (A,B) Levels of dimer and monomer nNOS (A) and total nNOS level (B) after treatment with 1 μM erastin± 100 μM cystamine for 8 h. (C‒F) Levels of NO (C,D) and ROS (E,F) after treatment with 1 μM erastin± 100 μM cystamine for 8 h: fluorescence microscopy images (C,E) and quantitative intensity values (D,F). Scale bar: 45 μm. (G‒K) Levels of ROS (G,H) and lipid ROS (I,J) after treatment with 1 μM erastin± 100 μM cystamine for 8 h: flow cytometry data (G,I), quantitative intensity values (H,J), and confocal microscopy data (K). Scale bar: 50 μm. (L) Cell viability change after treatment with 1 μM erastin± 100 μM cystamine for 24 h. Data are presented as the mean±SE. n=3. * P<0.05, ** P<0.01 vs the control group.

Article Snippet: Mouse anti-nNOS and rabbit anti-PDI antibodies were obtained from Solarbio (Beijing, China).

Techniques: Fluorescence, Microscopy, Flow Cytometry, Confocal Microscopy, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Biochemical mechanism of erastin-induced ferroptotic cell death in neuronal cells

doi: 10.3724/abbs.2023058

Figure Lengend Snippet: LOC14 (a PDI inhibitor) protects cells against erastin-induced ferroptosis (A,B) Levels of dimer and monomer nNOS (A) and total nNOS (B) after treatment with 1 μM erastin± 0.5 μM LOC14 for 8 h. (C‒F) Levels of NO (C,D) and ROS (E,F) after treatment with 1 μM erastin± 0.5 μM LOC14 for 8 h: fluorescence microscopy images (C,E) and quantitative intensity values (D,F). Scale bar: 45 μm. (G‒K) Levels of ROS (G,H) and lipid ROS (I,J) after treatment with 1 μM erastin± 0.5 μM LOC14 for 8 h: flow cytometry data (G,I), quantitative intensity values (H,J), and confocal microscopy data (K). Scale bar: 50 μm. (L) Cell viability change after treatment with 1 μM erastin± 0.5 μM LOC14 for 24 h. Data are presented as the mean±SE. n=3. * P<0.05, ** P<0.01 vs the control group.

Article Snippet: Mouse anti-nNOS and rabbit anti-PDI antibodies were obtained from Solarbio (Beijing, China).

Techniques: Fluorescence, Microscopy, Flow Cytometry, Confocal Microscopy, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Biochemical mechanism of erastin-induced ferroptotic cell death in neuronal cells

doi: 10.3724/abbs.2023058

Figure Lengend Snippet: A schematic depiction of the proposed role of PDI in mediating chemically induced ferroptosis Our earlier study showed that PDI in untreated HT22 cells is mostly present in its S-nitrosylated form, and GSH depletion is associated with S-denitrosylation of PDI [22]. Under conditions of cellular GSH depletion ( i. e., with an increased GSSG/GSH ratio), the reduced form of PDI is facilely converted to its oxidized form by ERO1. Oxidized PDI is the active form for catalyzing the conversion of the nNOS monomer to its dimer form, which then leads to increased formation of NO, followed by accumulation of cellular ROS and lipid ROS and ultimately ferroptotic cell death.

Article Snippet: Mouse anti-nNOS and rabbit anti-PDI antibodies were obtained from Solarbio (Beijing, China).

Techniques: