Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 3. Decreased glutathione pools by CHAC1 reduces protein-SSG and aggravates APAP-induced hepatotoxicity and ferroptosis in APAP-injured mice liver. (A) Quantification of GSH in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (B) Quantification of GSSG in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad- CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (C) Western blot analysis of S-glu tathionylated proteins in the liver tissues of Chac1+/+ Ad-GFP and Chac1−/−Ad-GFP mice treated with 300 mg/kg APAP for 2 and 6 h. GAPDH was used as an internal reference, followed by quantification of relative levels of S-glutathionylated proteins after 6 h of APAP treatment (Data are mean ± SEM of n = 3 mice/ group, t-test). (D) Western blot analysis of S-glutathionylated proteins and CHAC1-FLAG protein in the liver tissues of Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. GAPDH was used as an internal reference. Statistical chart shows the relative expression levels of S-glutathionylated proteins 6 h after APAP treatment (Data are mean ± SEM of n = 4 mice/group, t-test). (E) Serum levels of ALT and AST in Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h (Data are mean ± SEM of n = 5 mice/group, t-test). (F) H&E staining, 4-HNE protein adduct staining in liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. Scale bars = 200 μm. The area of liver injury and immunohistochemical score for 4-HNE protein adduct staining were quantified (Data are mean ± SEM of n = 5 mice/group, t-test). APAP, acetaminophen; GSH, glutathione; GSSG, oxidized glutathione; ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, haematoxylin and eosin; Chac1−/−, Chac1- deficient; Chac1+/+, wild-type controls.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Saline, Western Blot, Expressing, Staining, Immunohistochemical staining

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 4. Redox proteomic analysis reveals that the S-glutathionylation of Cys90 on ARF6 is regulated by CHAC1 in ferroptotic PMHs induced by APAP. (A) Flowchart outlining the key experimental procedures for proteomic analysis of S-glutathionylation. (B) Scatter plot illustrating the distribution of differential modification sites, sorted by the ratio of Ad-GFP + APAP/Ad-GFP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences, and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (C) Scatter plot showing the distribution of differential modification sites, sorted by the ratio of Ad-CHAC1 + APAP/Ad-GFP + APAP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (D) Venn diagram showing differentially modified sites under both APAP stimulation and CHAC1 overexpression (Fold change ≥1.2). (E) The heat map illustrating the union of differential modification sites in Ad-GFP, Ad-GFP + APAP, Ad-CHAC1, and Ad-CHAC1 + APAP comparison groups (CV < 0.1, fold change ≥1.2). (F) Scatter plot showing differentially modified sites under both APAP stimulation and CHAC1 overexpression; the order was sorted by the ratio of Ad-GFP + APAP/Ad-GFP (CV < 0.1, fold change ≥1.2). (G) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in PMHs. The secondary mass spectrum shows fragment ion information of the ARF6 C90 peptide segment. (H) Histogram showing the relative modification abundance of ARF6 C90 in different treatment groups, with glutathionylated peptides identified and quantified by LC-MS/MS (All values were normalized by the mean of the AdGFP-CON group, data are mean ± SEM of n = 2 biologically independent samples). (I) IP assay showing the expression of S-glutathionylated ARF6 in 293T cells overexpressing Myc-tagged ARF6. Whole cell lysates were used to confirm the expression of ARF6. (J) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in 293T cells overexpressing Myc-tagged ARF6. The secondary mass spectrum shows fragment ion infor mation of the ARF6 C90 peptide segment. PMH, primary mouse hepatocyte; IP, immunoprecipitation.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Modification, Over Expression, Comparison, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Immunoprecipitation

Journal: Physiological Reports

Article Title: Endothelin inhibits renin release from juxtaglomerular cells via endothelin receptors A and B via a transient receptor potential canonical‐mediated pathway

doi: 10.14814/phy2.12240

Figure Lengend Snippet: RT‐PCR for ETA (top) and ETB (bottom) identifying bands at 260 and 231 bp, respectively. Column 1 is the “no‐template” negative control. Column 2 is total mRNA obtained from isolated juxtaglomerular (JG) cells. Column 3 is total mRNA obtained from the positive control of liver. Column 4 is the calibration scale (100 bp ladder).

Article Snippet: Afterward, slides were incubated for 1 h at 37°C with a 1:50 dilution of an antibody against mouse ETA or ETB receptor protein (Alomone, Jerusalem, Israel), and then for 1 h at 37°C with a 1:100 dilution of secondary antibody (Alexa Fluor 568 goat antimouse IgG; Life Technologies, Grand Island, NY).

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control, Isolation, Positive Control

Journal: Physiological Reports

Article Title: Endothelin inhibits renin release from juxtaglomerular cells via endothelin receptors A and B via a transient receptor potential canonical‐mediated pathway

doi: 10.14814/phy2.12240

Figure Lengend Snippet: Immunofluorescence of a single juxtaglomerular (JG) cell using two antibodies; one specific for renin (in green) to confirm this is a JG cell, and another specific for the ETA (top) and ETB (bottom) receptors isoforms (in red). The third panel shows both renin and A1R in the same JG cell (merged image).

Article Snippet: Afterward, slides were incubated for 1 h at 37°C with a 1:50 dilution of an antibody against mouse ETA or ETB receptor protein (Alomone, Jerusalem, Israel), and then for 1 h at 37°C with a 1:100 dilution of secondary antibody (Alexa Fluor 568 goat antimouse IgG; Life Technologies, Grand Island, NY).

Techniques: Immunofluorescence

Journal: Physiological Reports

Article Title: Endothelin inhibits renin release from juxtaglomerular cells via endothelin receptors A and B via a transient receptor potential canonical‐mediated pathway

doi: 10.14814/phy2.12240

Figure Lengend Snippet: Renin release from juxtaglomerular (JG) cells under basal conditions (Control), after incubation with ET‐1, or ET‐1 plus different concentrations of the ETB receptor antagonist BQ788 (3 μ mol/L and 10 μ mol/L). Incubation with ET‐1 reduced renin release. This response was inhibited by 10 μ mol/L of an ETB receptor blocker. * P < 0.05 versus control.

Article Snippet: Afterward, slides were incubated for 1 h at 37°C with a 1:50 dilution of an antibody against mouse ETA or ETB receptor protein (Alomone, Jerusalem, Israel), and then for 1 h at 37°C with a 1:100 dilution of secondary antibody (Alexa Fluor 568 goat antimouse IgG; Life Technologies, Grand Island, NY).

Techniques: Incubation