Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: ( A ) Recombinant Trx1 was S-nitrosylated using GSNO in vitro. The NO group was replaced with biotin-HPDP in biotin switch assays. LC-MS/MS analyses were performed. The MS/MS spectrum of m / z 789.36 resulting from a doubly charged ion ( m / z 788.86) with an isotope space (0.5) corresponds to the peptide sequence of 73 CMPTFQFFK 81 with a biotin-HPDP (+428.19 Da) modification on Cys73 in Trx1. The b- and y-ion series correspond to the fragment ions of peptides from the N- and C-terminus, respectively, which confirms the peptide sequence of Trx1(73–81). The b-ion series (from the N-terminus), but not y-ion series (from the C-terminus), correspond to the fragment ions with a biotin-HPDP, indicating covalent binding of biotin-HPDP to Cys73. ( B ) Cys73 of Trx1 is S-nitrosylated. Cardiomyocytes were transduced with Ad-LacZ, Ad-Flag-Trx1 WT-HA, or Ad-Flag-Trx1(C73S)-HA for 48 hours. A biotin switch assay was performed, followed by pull-down with streptavidin-agarose to detect S-nitrosylated Trx1. The ratio of S-nitrosylated Trx1/total Trx1 is shown. * P < 0.05 vs. Trx1 WT, n = 3. Error bars represent SEM. ( C ) Cys73 is conserved among vertebrates. ( D ) Trx1 is oxidized during GD. Cardiomyocytes were lysed with biotin-labeled iodoacetamide (BIAM) after indicated periods of GD. The BIAM-labeled reduced form of Trx1 was pulled down with streptavidin-agarose. n = 6. ( E ) Trx1 is S-nitrosylated in response to GD in cardiomyocytes. SNO-Trx1 was detected by biotin switch assay. n = 5. ( F ) Trx1 is S-nitrosylated in response to GD in cardiac fibroblasts. ( G ) Cys73 of Trx1 promotes cell survival under GD conditions. Cardiomyocytes were transduced with lacZ , Flag-Trx1 WT-HA, Flag-Trx1(C73S)-HA, and shTrx1 adenoviruses for 48–96 hours, after which they were incubated with normal or glucose-free medium for 24 hours. Cell death was assessed using CellTiter-Blue. n = 12. * P < 0.05 by 2-tailed Student’s t test ( B , E , and F ) or 1-way ANOVA ( D and G )

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques: Recombinant, In Vitro, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing, Modification, Binding Assay, Transduction, Biotin Switch Assay, Labeling, Incubation

Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: ( A ) Cardiomyocytes were transduced with the indicated adenovirus along with Ad-mRFP-GFP-LC3 for 48 hours. Cardiomyocytes were cultured in normal or glucose-free medium for 4 hours. Autophagosomes and autolysosomes (yellow and free red puncta) were counted and quantified. n = 3. ( B ) Autophagic flux was assessed using Ad-GFP-LC3-RFP. Indicated adenovirus vectors were transduced into cardiomyocytes. The cells were incubated with glucose-free medium for 4 hours. The GFP/RFP ratio is shown. n = 3. Scale bar: 20 μm. ( C ) Trx1-C73S–KI mice exhibited normal cardiac function under basal and starvation conditions. n = 8. ( D ) Trx1 Cys73 mediates starvation-induced autophagy in the heart. LC3-II formation was evaluated after 48 hours of starvation with chloroquine (CQ) treatment. n = 6–8. ( E ) Trx1-C73S–KI mice and WT mice were subjected to ischemia for 3 hours. Representative images of TTC staining are shown. Scale bar: 5 mm. The percentage infarct area/area at risk (AAR) and AAR (%) are shown. ( F ) Echocardiographic measurements after 3 hours of ischemia. n = 5–6. ( G ) Plasma troponin T levels were evaluated after 3 hours of ischemia. n = 4–5. Error bars represent SEM. * P < 0.05; ** P < 0.01 by 1-way ANOVA ( A – D and F ) or 2-tailed Student’s t test ( E and G ).

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques: Transduction, Cell Culture, Incubation, Staining, Clinical Proteomics

Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: ( A ) Cardiomyocytes were transduced with Ad-Flag-Trx1 C35S and Ad-Atg7 for 48 hours and then treated with 100 μM H 2 O 2 for 30 minutes. Interaction between Trx1 and Atg7 was examined. ( B ) Cardiomyocytes were transduced with Ad-Flag-Trx1 C35S and Ad-Atg7 for 48 hours and then interaction between Trx1 and Atg7 was examined in the presence or absence of DTT. ( C ) Tg-Flag-Trx1 C35S and WT mice were subjected to sham operation or ischemia for 20 minutes. Homogenates were prepared from sham/ischemic areas. Coimmunoprecipitation with anti-FLAG–agarose beads followed by immunoblotting for Atg7 was performed. Representative immunoblots are shown. n = 4. ( D ) Cardiomyocytes were treated with the indicated concentrations of H 2 O 2 for 10 minutes and labeled with biotin-labeled iodoacetamide (BIAM) upon lysis. Atg7 with reduced cysteines was recovered with streptavidin-agarose. n = 3. * P < 0.05 by 1-way ANOVA ( C ) or Kruskal-Wallis test ( D ). ( E ) Evolutionarily conserved Cys545 and Cys548 form an intramolecular disulfide bond. Intramolecular disulfide bonds were identified by MS analysis using recombinant Atg7. Evolutionary conservation of Cys545 and Cys548 is shown (upper panel). Intramolecular disulfide bonds are indicated by red lines (lower panel). ( F ) HEK293 cells were transduced with the indicated plasmids for 48 hours and then interaction between Trx1 and Atg7 was examined. ( G ) Atg7-KO AMCMs were transduced with Ad-LacZ, Ad-Atg7 WT, or Ad-Atg7 CC545/548SS for 24 hours. Protein samples were prepared and Western blotting was performed to detect LC3 and Atg7.

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques: Transduction, Western Blot, Labeling, Lysis, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: ( A and B ) Trx1 promotes GD-induced Atg7 oxidation. Cardiomyocytes were transduced with Ad-shTrx1 ( A ) or Ad-Trx1 ( B ). The redox status of Atg7 was examined with BIAM pulldown. n = 6–10 ( A ) and 6 ( B ). ( C ) Atg7 prevents GD-induced Trx1 oxidation in a Cys545-Cys548–dependent manner. Cardiomyocytes were transduced with Ad-Atg7 or Ad-Atg7(CC545/548SS). The redox status of Trx1 was examined with BIAM pulldown. n = 3–6. ( D and E ) Atg7 reduces Trx1 in an in vitro reconstitution system. ( D ) Recombinant Atg7 was reduced with DTT and dialyzed to remove DTT. Reduced Atg7 was incubated with oxidized Trx1. Reduced Trx1 was detected with BIAM labeling. n = 5. ( E ) The redox status of Atg7 at Cys545-Cys548 and Trx1 at Cys32-Cys35 was examined with MS analyses. Experiments were conducted in duplicate. CBB, Coomassie brilliant blue. * P < 0.05 by 1-way ANOVA ( A – D ).

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques: Transduction, In Vitro, Recombinant, Incubation, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: ( A – E ) S-nitrosylation of Atg7 was analyzed with biotin switch assays. ( A ) Trx1 mediates GSNO-induced S-nitrosylation of Atg7. Cardiomyocytes were treated with 100 μM GSNO for 30 minutes. n = 4. ( B ) WT mice were subjected to either sham operation or ischemia for 30 minutes. n = 6. ( C ) WT and cardiac-specific heterozygous Trx1-KO mice were subjected to ischemia for 30 minutes. n = 5–6. ( D ) The level of SNO-Atg7 was examined in homozygous Trx1-C73S–KI mice. n = 6. ( E ) Atg7-KO AMCMs were transduced with Ad-LacZ, Ad-Atg7 WT, or Ad-Atg7 CC545/548SS for 24 hours. Biotin switch assays were performed to detect SNO-Atg7. n = 5. ( F ) Trx1 regulates Atg7 in a Cys73-dependent manner. Conjugation of Atg5 and Atg12 was assessed in Trx1-C73S–KI mice after 48 hours of starvation (Stv). n = 6. ( G ) Possible S-nitrosylation substrates of Trx1 in the heart. Proteins whose S-nitrosylation is changed in Trx1-C73S–KI mice were identified with biotin switch assays followed by MS identification. ( H ) Fhl2 reduces Trx1 in an in vitro reconstitution system. Recombinant His-tagged Fhl2 was immobilized with Ni-NTA resin and reduced with DTT. To remove DTT, Ni-NTA resin was washed with 1 mL buffer twice. Fhl2 was then incubated with oxidized Trx1. Reduced Fhl2 and Trx1 were labeled with BIAM. n = 4. * P < 0.05 by 1-way ANOVA ( A , C , D , F , and H ) or 2-tailed Student’s t test ( B , E , and G ).

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques: Transduction, Conjugation Assay, In Vitro, Recombinant, Incubation, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: ( A ) Rat cardiomyocytes transduced with Ad-Atg7 were cultured in normal or glucose-free medium for 4 hours. A biotin switch assay was performed, followed by MS analysis to determine sites of S-nitrosylation in Atg7. ( B ) Recombinant Trx1 treated with GSNO was incubated with recombinant human Atg7 protein for 30 minutes. A biotin switch assay followed by MS analysis was performed to determine sites of transnitrosylation in Atg7. A representative MS spectrum is shown. ( A and B ) The MS/MS spectra of the peptides 283 LPEMAFSPDC*PK 296 and 394 QPLYEFEDC*LGGGKPK 409 with a biotin-HPDP (+428.19 Da) modification on C294 and C402 are shown. The MS/MS spectrum of m / z 881.9 corresponds to the peptide sequence of Atg7 (283–296) with a biotin-HPDP. That of Atg7 (394–409) with a biotin-HPDP was not detected due to complete fragmentation of the precursors. The y- and b-ion series confirmed the peptide sequence and the biotin-HPDP modification on the cysteines. The rat Atg7 peptides 283–296 and 394–409 correspond to human Atg7 289–300 and 398–413, respectively. ( C ) Cardiomyocytes were transduced with Ad-LacZ, Ad-Trx1 WT, or Ad-Trx1(C73S). A biotin switch assay followed by Western blotting was carried out to measure S-nitrosylation of Atg7. Representative immunoblots and quantification analysis of S-NO Atg7/total Atg7 are shown. n = 3. ( D ) Cardiomyocytes were transduced with Ad-Atg7 WT or Ad-Atg7 CC294/402SS in the presence of Ad-LacZ or Ad-Trx1 WT. A biotin switch assay followed by Western blotting was performed to measure S-nitrosylation of Atg7. Representative immunoblots and quantification analysis of SNO-Atg7/total Atg7 are shown. n = 3. * P < 0.05 by Kruskal-Wallis test ( C ) or 2-tailed Student’s t test ( D ).

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques: Transduction, Cell Culture, Biotin Switch Assay, Recombinant, Incubation, Tandem Mass Spectroscopy, Modification, Sequencing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

doi: 10.1172/JCI162326

Figure Lengend Snippet: Trx1 is oxidized at Cys32-Cys35 in response to GD, which is prerequisite for S-nitrosylation at Cys73. Cys32-Cys35 are reduced by Cys545-Cys548 of Atg7, which triggers S-nitrosylation of Atg7 at Cys402. S-nitrosylation of Atg7 promotes autophagy.

Article Snippet: Antibodies against the following proteins were used in this study: Trx1 (Cell Signaling Technology [CST], 2429), LC3 (MBL Intl.

Techniques:

Journal: Oncogene

Article Title: Thioredoxin participates in a cell death pathway induced by interferon and retinoid combination.

doi: 10.1038/sj.onc.1204477

Figure Lengend Snippet: Figure 5 Thioredoxin reductase activity in cells expressing various Trx1 mutants. Data indicates mean+s.e. of triplicates. In (a), the basal TR activity using endogenous Trx was measured using 50 mg of total cell extract. In (b), the catalytic activity of TR in the extracts of various cell lines was measured using exogenous Trx as substrate. In each case background enzymatic activity (in the absence of exogenous Trx) was measured and subtracted from the experimental values. In this experiment 20 mg of cell extract was used. Each bar represents mean+s.e. of triplicates. The absorbance was in the linear range compared to control enzyme, TR

Article Snippet: Restriction and DNA modifying enzymes (NE Biolabs); G418 Sulfate, IPTG and Lipofectamine plus (Life Technologies); nitrocellulose membranes, ECL reagents and horseradish peroxidase coupled to anti-rabbit or anti mouse antibodies (Amersham Pharmacia Inc); human IFN-bser (Berlex Inc.), mouse monoclonal antibodies against actin (Sigma Inc.) and thioredoxin (Serotec Inc.) and rabbit polyclonal antibodies against caspase-8; polyADP ribose polymerase (Santa Cruz Biotech Inc.) were employed in these studies.

Techniques: Activity Assay, Expressing, Control

Journal: Advanced Science

Article Title: Identification of PRDX5 as A Target for The Treatment of Castration‐Resistant Prostate Cancer

doi: 10.1002/advs.202304939

Figure Lengend Snippet: Upregulation of the thioredoxin/peroxiredoxin pathway in DTP cells A) LNCaP epiDTP or enzDTP cells were treated for 48 h with EPI001 (EPI 50 µM) or enzalutamide (ENZ 60 µM), respectively, plus each ninety‐three drugs (10 µM) identified in Figure . Experiments identified 9 drugs that reduced cell survival below 70%. Each data point represents the average of three independent experiments and is expressed as a percentage of viable cells relative to untreated controls as measured by CCK‐8 assay. Standard deviations are less than 5%. B) The highest concentration that does not affect LNCaP cell viability (IC 0 ) was determined for the above 9 drugs. Subsequently, LNCaP epiDTP or enzDTP cells were treated with each drug (IC 0 ) with or without EPI001 (EPI 50 µM) or enzalutamide (ENZ 60 µM) for 48 h. Additive or synergistic effects were seen for all nine drugs. Each data point represents the average of three independent experiments and is expressed as a percentage of viable cells relative to untreated controls as measured by CCK‐8 assay. Standard deviations are less than 5%. C) Combination indexes (CI) of nine drugs were measured. A CI of less than, equal to, and more than 1 indicates synergy, additivity, and antagonism, respectively. D) 22Rv1 cells that are resistant to ENZ were treated for 48 h with ENZ (60 µM), each of the 9 drugs identified above, or in combinations. Results indicated that all 9 drugs either alone or in combination with ENZ could reduce cell survival. Each data point represents the average of three independent experiments and is expressed as a percentage of viable cells relative to untreated controls as measured by CCK‐8 assay. Standard deviations are less than 5%. E) Diagram portrays the thioredoxin/peroxiredoxin pathway and up‐regulation of key proteins (red color) in DTP cells. F: Confirmation of proteomics results (TXNRD1, TXNRD2, TXNRD3, TXN2, and PRDX5) by Western blotting. GAPDH protein is used as the loading control. Data are expressed as mean ± std of triplicates. One‐way ANOVA with the Turkey test was performed. p <0.05 was considered significant and indicated by different letters.

Article Snippet: PRDX5 (67599‐1‐Ig; 1:2000), CDKN1A (p21) (60214‐1‐Ig; 1:1000), Cyclin E1 (CCNE1) (11554‐1‐AP; 1:1000), Cyclin B1 (CCNB1) (67686‐1‐Ig; 1:5000), CDC6 (11640‐1‐AP; 1:2000), CDC2 (CDK1) (19532‐1‐AP; 1:1000), TXNRD1 (67728‐1‐Ig; 1:10 000), TXNRD2 (16360‐1‐AP; 1:3000), TXNRD3 (19517‐1‐AP; 1:2000), TXN2 (13089‐1‐AP; 1:1000), NSE (66150‐1‐Ig; 1:10 000), CgA (60135‐2‐Ig; 1:3000), CgB (14968‐1‐AP; 1:2000), PTHrP (29115‐1‐AP; 1:800), AMACR (15918‐1‐AP; 1:500), GAPDH (60004‐1‐Ig; 1:50 000), β‐ACTIN (66009‐1‐Ig; 1:10 000) antibodies were purchased from Proteintech Group (IL, US). β‐TUBLIN (86 298; 1:10 000) antibody was purchased from Cell Signaling Technology (MA, US).

Techniques: CCK-8 Assay, Concentration Assay, Western Blot, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: The thioredoxin reductase inhibitor BS suppressed lung cancer in vitro and in vivo. (a) Molecular structure of butaselen. (b) BS sensitivity screen in lung cancer cells and non-transformed cell lines. IC50s determined by growth assays using CCK-8 assay. (c) Viability of A549 and H1299 cells by BS treatment. Cells were treated with BS (0, 5, 10, 15, 20, 30 μM) for 24, 48 h, or 72 h, followed by CCK-8 assay. Error bars are means ± std (n = 3 in A549, n = 4 in H1299). (d) Crystal violet staining of A549 and H1299 cells. Cells were treated with BS (0, 5, 10, 20, 30 μM) for 24 h, followed by a consecutive culture of 7∼14 days before stained by crystal violet. Error bars are means ± std. * P < 0.05, *** P < 0.001, compared with the control group by One-way ANOVA (n = 3). (e) Tumor volume of LLC1-bearing mice treated with BS and saline. The tumor volume was determined every two to five days during the experiment on LLC1-nodulated C57BL/6J mice. Error bars are means ± std. ** P < 0.01, analyzed by using Student’s t test (n = 6 mice/group). (f) Tumor weight of LLC1-bearing mice treated with BS and saline. Tumor weights were measured after the mice were sacrificed. Shown are the representative tumor images of mice after treatment with BS for 15 days. Error bars are means ± std. ** P < 0.01, analyzed by using Student’s t test (n = 6 mice/group). (g) Cellular TrxR activity of A549 and H1299 cells by BS treatment. Cells were treated with BS (0, 5, 10, 20, 30 μM) for 24 h, followed by the detection of TrxR activity. Error bars are means ± std. * P < 0.05, ** P < 0.01, **** P < 0.0001, compared with the control group by One-way ANOVA (n = 3). (h) Western blot of TrxR and Trx expression of A549 and H1299 cells treated with BS (0, 5, 10, 20, 30 μM) for 24 h. Error bars are means ± std. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the control group by One-way ANOVA (n = 3). (i) TrxR activity of tumor tissues. Error bars are means ± std. * P < 0.05, analyzed by using Student’s t test (n = 6 mice/group). (j) The expression of TrxR1 and Trx1 in tumor tissues was determined by western blot. Error bars are means ± std. * p < 0.05, ** p < 0.01, analyzed by using Student’s t test (n = 6 mice/group). (k) Cytotoxicity of BS on human organoid. Organoids were plated in 96-well plates and were treated in triplicate with BS at the indicated concentrations. CellTiter-Glo assay was applied for quantification of total ATP content. Error bars are means ± std. *** P < 0.001, **** P < 0.0001, compared with the control group by One-way ANOVA (n = 3). (l) Representative bright-field images of organoids treated by different concentration of BS (0, 5, 10, 20, 40 μM) for 24 h. Scale bar: 200 μm.

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: In Vitro, In Vivo, Transformation Assay, CCK-8 Assay, Staining, Control, Saline, Activity Assay, Western Blot, Expressing, Glo Assay, Concentration Assay

Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: The Trx system inversely associates with HBP1 in lung cancer. (a–d) The differential expression of TrxR1 (TXNRD1) (a), Trx1 (TXN) (b), TXNIP (TXNIP) (c), HBP1 (HBP1) (d) between normal (blue) and tumor samples (red) was analyzed by using GEPIA database ( http://gepia.cancer-pku.cn ). LUAD: lung adenocarcinoma. LUSC: Lung squamous cell carcinoma. (e) Expression of TrxR1, Trx1, TXNIP, and HBP1 proteins in adjacent normal (N) and tumor (T) tissues from lung cancer patients by western blot. Error bars are means ± std. * P < 0.05, ** P < 0.01, *** P < 0.001, analyzed by using Student’s t test (n = 10). (f) TrxR1 activity of adjacent normal and tumor tissues from lung cancer patients. Error bars are means ± std. * P < 0.05, analyzed by using Student’s t test (n = 10). (g–j) Kaplan-Meier survival curves indicating the correlation of TXNRD1 (g), TXN (h), TXNIP (i), and HBP1 (j) mRNA expression with the survival of LUAD and LUSC patients was analyzed by using the Kaplan Meier plotter database ( https://kmplot.com/analysis/index.php?p = home ). K-L The association between the mRNA levels of HBP1 and TXN (K), HBP1 and TXNIP (L) were conducted by employing Pearson correlation to calculate the correlation scores of genes by using GEPIA database ( http://gepia.cancer-pku.cn ).

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: Quantitative Proteomics, Expressing, Western Blot, Activity Assay

Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: TrxR1/Trx1 inhibition leads to reduced proteasomal degradation of HBP1. (a) A549 cells were transfected with control siRNA or siTrx1 for 48 h, then treated with BS (10 μM) or DMSO for another 24 h, followed by ROS detection with the fluorescent probe DCFH-DA. Error bars are means ± std. * P < 0.05, ** P < 0.01, compared with each indicated group by One-way ANOVA (n = 3). (b) A549 cells were transfected with control siRNA or siTrx1 for 48 h, then treated with BS (10 μM) or DMSO for another 24 h, followed by immunoblots. Error bars are means ± std. * P < 0.05, ** P < 0.01, **** P < 0.0001, compared with each indicated group by One-way ANOVA (n = 3). (c) A549 cells were transfected with control siRNA or siTrx1 for 48 h, then treated with BS (10 μM) or DMSO for another 24 h, followed by and Real-time PCR. Error bars are means ± std, compared with each indicated group by One-way ANOVA (n = 3). (d) A549 cells were treated with DMSO or BS (10 μM) for 24 h, followed by a further incubation with the protein translation inhibitor cycloheximide (CHX) for 0, 30, 60, 90 or 120 min before harvest. HBP1 protein levels were detected by western blot. β-actin was used as the loading control for this turnover experiment. Densitometry is plotted for the means ± std of three independent experiments. * P < 0.05, analyzed by using Student’s t test. (d) A549 cells were treated with BS (10 μM) for 24 h, followed by incubation with (lane 3 and 4) or without (lane 1 and 2) MG132 for another 6 h before harvest. HBP1 protein was detected by western blot. (f) HEK-293 T cells were co-transfected with Flag-HBP1, HA-Ub or siTrx1 for 48 h, followed by (co-)treatment with BS/NAC for 24 h and then exposed to 10 µM MG132 for another 6 h prior to lysis. HBP1 protein was then isolated by immunoprecipitation and analyzed by anti-Flag and anti-HA antibody. (g) A549 cells were lysed with IP lysis buffer and then subjected to immunoprecipitation with anti-HBP1 or anti-Trx1 antibodies followed by western blot analysis. (h) A549 cells were transfected with Flag-HBP1, with (co-)treatment of siTrx1, BS (10 μM), or NAC (5 mM). IP assay was carried out by using anti-Flag antibody and followed by western blot with anti-Flag/Trx1 antibody.

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: Inhibition, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Lysis, Isolation, Immunoprecipitation

Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: TrxR/Trx inhibition enhances the transcriptional activity of HBP1 on DNMT1. (a) A549 cells were transfected with control siRNA or siTrx1 for 48 h, then treated with BS (10 μM) or DMSO for another 24 h, followed by Real-time PCR detecting the mRNA level of DNMT1. Error bars are means ± std. * P < 0.05, **** P < 0.0001, compared with each indicated group by One-way ANOVA (n = 3). (b) HEK293 T cells were co-transfected with 0.1 μg of the indicated reporters (native Luc-DNMT1 and mutant Luc-ΔDNMT1 promoters) and siTrx1 or BS (10 μM). The luciferase activities were expressed as the means ± std from three experiments. Statistical differences were analyzed using One-way ANOVA. * P < 0.05, *** P < 0.001. (c) HEK-293 T cells were transfected with control siRNA or siTrx1 for 48 h, then (co-)treated with BS (10 μM) for another 24 h, followed by ChIP assay with anti-HBP1 antibody. Error bars are means ± std. * P < 0.05, compared with each indicated group by one-way ANOVA (n = 3). (d) The global DNA methylation levels of the genome DNA from tumor tissues of mice in the control or BS group were detected by ELISA. Error bars are means ± std. **** P < 0.0001, compared with the control group by Student’s t test (n = 6). (e) The DNA methylation levels of the CG sites on the p21 promoter from tumor tissues of mice in the control or BS group were detected by bisulfite PCR. The heatmap showed the percentage of methylation of 9 CpGs on the promoter. The scatter plots showed the average percentage of methylation of these 9 CpGs. Error bars are means ± std. ** P < 0.01, compared with the control group by Student’s t test (n = 3). (f) The DNA methylation levels of the CG sites on the HOXA9 promoter from tumor tissues of mice in the control or BS group were detected by bisulfite PCR. The heatmap showed the percentage of methylation of 10 CpGs on the promoter. The scatter plots showed the average percentage of methylation of these 10 CpGs. Error bars are means ± std. * P < 0.05, compared with the control group by Student’s t test (n = 3). (g) A549 cells were transfected with control siRNA or siTrx1 for 48 h, then treated with BS (10 μM) or DMSO for another 24 h, followed by Real-time PCR detecting the mRNA level of p21. Error bars are means ± std. ** P < 0.01, **** P < 0.0001, compared with each indicated group by One-way ANOVA (n = 3). (h) A549 cells were transfected with control siRNA or siTrx1 for 48 h, then treated with BS (10 μM) or DMSO for another 24 h, followed by Real-time PCR detecting the mRNA level of HOXA9. Error bars are means ± std. * P < 0.05, ** P < 0.01, **** P < 0.0001, compared with each indicated group by One-way ANOVA (n = 3).

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: Inhibition, Activity Assay, Transfection, Control, Real-time Polymerase Chain Reaction, Mutagenesis, Luciferase, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Methylation

Journal: Redox Report : Communications in Free Radical Research

Article Title: The novel thioredoxin reductase inhibitor butaselen suppresses lung cancer by inducing oxidative stress

doi: 10.1080/13510002.2025.2588086

Figure Lengend Snippet: Schematic model of lung cancer inhibition by BS. The TrxR/Trx inhibitor butaselen (BS) can inhibit lung cancer by inducing ROS-dependent apoptosis. The inactivation of the NF-κB and PI3K-Akt signaling pathways, along with the activation of the p38/JNK signaling pathway, contributes to the anti-cancer effects of BS on lung cancer. Although p53 itself is not activated by BS, the HBP1/DNMT1/p21/γ-H2AX/Bcl-2/Bax signaling pathway is activated by BS and contributes to its tumor-inhibitory role. Further mechanistic studies revealed HBP1 as a novel target of the Trx system. The Trx system inversely associates with HBP1 in lung cancer and regulates HBP1 expression at the post-translational level. Under normal conditions, TrxR1 catalyzes the reduction of Trx1 by utilizing NADPH. In its reduced form, Trx1 interacts with HBP1, promoting the ubiquitination of HBP1, which leads to its proteasomal degradation and maintains a low level of HBP1 within cancer cells. Treatment with butaselen inhibits the activity of TrxR1 in lung cancer cells, resulting in the oxidation of Trx1 and the subsequent excessive generation of ROS. HBP1 is activated after being released by the oxidized Trx1 and escaping proteasomal degradation. The activated HBP1 inhibits the expression of DNMT1 and elevates Bax. The decreased DNMT1 further results in the demethylation of the whole genome as well as the promoters of p21 and HOXA9. Ultimately, the upregulation of p21 and γ-H2AX, along with the downregulation of DNMT1 and Bcl-2/Bax, contributes to the apoptosis of lung cancer cells induced by BS. Taken together, the TrxR/Trx inhibitor butaselen inhibits lung cancer by promoting ROS-induced apoptosis through the NF-κB, PI3K-Akt, p38/JNK, and HBP1/DNMT1 signaling pathways.

Article Snippet: The primary antibodies used for immunoblotting analysis are as follows: TrxR1 (Proteintech, 11117-1-AP), Trx1 (Proteintech, 14999-1-AP), HBP1 (Proteintech, 11746-1-AP), DNMT1 (Proteintech, 24206-1-AP), Bcl-2 (Proteintech, 12789-1-AP), Bax (Proteintech, 50599-2-Ig), β-actin (Bioss, bs-0061R), Flag (Sigma-Aldrich, F1804), HA (Covance, MMS-101P), p53 (Santa, sc-126), p21 (MBL, K0081-3), p27 (MBL, K0082), γ-H2AX (CST, 9718), NF-κB (Abcam, ab32536), p-NF-κB(CST, 3033), p38 (Santa, sc-7972), p-p38 (Santa, sc-101759), JNK (MCE, HY- P80728 ), p-JNK (Immunoway, YP0157), Akt (Santa, sc-5298), phospho-Akt (CST, 4060).

Techniques: Inhibition, Protein-Protein interactions, Activation Assay, Expressing, Ubiquitin Proteomics, Activity Assay

Journal: Journal of animal science and biotechnology

Article Title: Increased maternal consumption of methionine as its hydroxyl analog improves placental angiogenesis and antioxidative capacity in sows.

doi: 10.1186/s40104-025-01159-z

Figure Lengend Snippet: Fig. 7 Increased consumption of methionine as OHMet improved angiogenesis and antioxidative capacity in pTr cells. A The trophoblast cells were treated for 24 h with 20% serum from each group (n = 3). B Cell viability. C Reactive oxidative species level. D Schematic of the Met metabolism and list of genes directly involved and associated with this pathway. E The gene expression of transmethylation and trans-sulphuration. F The gene expression of anti-oxidation. G The gene expression of angiogenesis. E–G Duplicated independent experiments. H The protein expression of pTr, GAPDH as a loading control. TXN, thioredoxin; GSTT1, glutathione transferase T1; VEGF-A, vascular endothelial growth factor A. Significant difference among four treatments is marked with different superscripts (P < 0.05). Abbreviations: CON = basal diet; 1.5S-OHMet = basal diet + 1.5 g/kg OHMet; 3.0S-OHMet = basal diet + 3.0 g/kg OHMet; 3.0S-Met = basal diet + 3.0 g/kg Met

Article Snippet: Primary antibodies were thioredoxin (TXN, Zen BioScience, 1:1,000), HADHA (Zen BioScience, 1:1,000), NDUFA3 (Zen BioScience, 1:1,000), ENO3 (Zen BioScience, 1:1,000), GSTT1 (Proteintech, 1:1,000), GAPDH (Absin, 1:1,000), β-actin (Cell Signaling Technology, 1:1,000).

Techniques: Gene Expression, Expressing, Control

Journal: Journal of animal science and biotechnology

Article Title: Increased maternal consumption of methionine as its hydroxyl analog improves placental angiogenesis and antioxidative capacity in sows.

doi: 10.1186/s40104-025-01159-z

Figure Lengend Snippet: Fig. 8 Anti-proliferative and inhibited antioxidant capacity induced by homocysteine in pTr cells (n = 3). A Detection of LDH activity in pTr cells. B EdU-positive cells were measured using ImageJ software. C Cell proliferation was determined by the EdU assay, and images were taken under a fluorescence microscope (magnification 400 × , bar = 100 μm). D Cell migration was measured using ImageJ software. E The cell migration distance (bar = 500 μm) and images of ROS were taken using a DCFH-DA probe (magnification 400 × , bar = 500 μm). F Representative western blot results for protein levels of enolase 3 (ENO3) and thioredoxin (TXN), β-actin was used as a loading reference. G–J Detection of apoptosis in pTr cells. Annexin V and propidium iodide (PI) fluorescence was quantified using flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary antibodies were thioredoxin (TXN, Zen BioScience, 1:1,000), HADHA (Zen BioScience, 1:1,000), NDUFA3 (Zen BioScience, 1:1,000), ENO3 (Zen BioScience, 1:1,000), GSTT1 (Proteintech, 1:1,000), GAPDH (Absin, 1:1,000), β-actin (Cell Signaling Technology, 1:1,000).

Techniques: Activity Assay, Software, EdU Assay, Fluorescence, Microscopy, Migration, Western Blot, Flow Cytometry