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: 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

Journal: Antioxidants

Article Title: Ponicidin Inhibits Lung Cancer Progression Through Coordinated Downregulation of Sulfhydryl Antioxidants and TrxR1

doi: 10.3390/antiox15010100

Figure Lengend Snippet: Ponicidin affects cellular oxidative stress-related pathway in lung cancer cells. ( A ) The Venn diagram illustrates 12 common differentially expressed genes between ponicidin-treated and -untreated H1299 and H1975 cells. ( B ) Pathway enrichment analyses were performed on the 12 differentially expressed genes to elucidate their biological functions. ( C ) A molecular docking study was conducted to evaluate the binding potential of ponicidin toTrxR1, identifying critical amino acids involved in this interaction. ( D ) Western blot assay showing the expression of the antioxidant proteins (PRDX1, TrxR1, and Trx1) in lung cancer cells treated with ponicidin (0, 10, 20, 30 μM) for 24 h.

Article Snippet: Antibodies targeting PRDX1 (#8499) and GAPDH (#5174), and TrxR1 (#15140) were sourced from Cell Signaling Technology (Shanghai, China), while ABclonal (Wuhan, China) provided the antibodies against Trx1 (A7638).

Techniques: Binding Assay, Western Blot, Expressing

Journal: Technology in cancer research & treatment

Article Title: Human papillomavirus16 E6 but not E7 upregulates GLUT1 expression in lung cancer cells by upregulating thioredoxin expression.

doi: 10.1177/15330338211067111

Figure Lengend Snippet: Figure 2. Effects of E6 on the regulation of Trx, p65 and GLUT1 expression via (A) transfection and (B) siRNA transfection. Effect of E7 on the regulation of Trx expression via (C) transfection and (D) siRNA transfection. Western blot and reverse transcription-quantitative PCR analyses were performed to detect E6, Trx, p65, and GLUT1 expression levels in the lung cancer cell lines. *P < .05, **P < .01. Trx, thioredoxin; si, small interfering; NS: nonspecific siRNA.

Article Snippet: HPV16 E6 small interfering (si) RNA and HPV16 E7 siRNA were purchased from Guangzhou RIBOBIO Co., Ltd, while Trx siRNA and p65 siRNA were purchased from Santa Cruz Biotechnology, Inc. Disordered siRNA was used as a nonspecific siRNA control.

Techniques: Expressing, Transfection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Technology in cancer research & treatment

Article Title: Human papillomavirus16 E6 but not E7 upregulates GLUT1 expression in lung cancer cells by upregulating thioredoxin expression.

doi: 10.1177/15330338211067111

Figure Lengend Snippet: Figure 3. Effects of Trx on the regulation of p65 and GLUT1 expression via (A) transfection and (B) siRNA transfection. Effect of p65 on the regulation of GLUT1 expression via (C) siRNA transfection. Western blot and reverse transcription-quantitative PCR analyses were performed to detect Trx, p65, and GLUT1 expression levels in lung cancer cell lines. *P < .05, **P < 0.01. Trx, thioredoxin; si, small interfering; NS, nonspecific siRNA.

Article Snippet: HPV16 E6 small interfering (si) RNA and HPV16 E7 siRNA were purchased from Guangzhou RIBOBIO Co., Ltd, while Trx siRNA and p65 siRNA were purchased from Santa Cruz Biotechnology, Inc. Disordered siRNA was used as a nonspecific siRNA control.

Techniques: Expressing, Transfection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: HLA-DR cancer cells expression correlates with T cell infiltration and is enriched in lung adenocarcinoma with indolent behavior

doi: 10.1038/s41598-021-93807-3

Figure Lengend Snippet: Mass cytometry antibody panel for lung adenocarcinoma.

Article Snippet: Thioredoxin , 146-Nd , Intracellular , 2G11/TRX , Fluidigm , 3146016B.

Techniques: Mass Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 1. Trx1 is present on the cell surface of HUVEC in a reduced form and limits MAC deposition. (A) Detection of Trx1 on HUVEC by flow cytometry using rabbit anti-Trx1 serum (solid line) and preimmune serum from the same animal as a negative control (broken line). A number denotes percentage of cells staining positive for Trx1, gated above the 95th percentile of the negative control. (B) Western blots of HUVEC lysates after incubation of cells with PBS or non–membrane-permeable bIAA or bNHS to label surface proteins. Lysates were blotted for Trx1 (left panel) or biotin (right panel). (C) Western blot for Trx1 after use of neutravidin beads to precipitate biotinylated proteins from HUVEC lysates after cells were incubated with either PBS or bIAA. Each experiment was performed independently three times with similar results. (D) Trx1 activity can be assessed using an insulin assay, where in the presence of DTT, Trx1 causes reduction of insulin and subsequent aggregation of the liberated insulin b-chain, measurable by absorbance at 650 nm. The addition of Trx-specific F(ab)2 (broken lines), but not nonspecific F(ab)2 (solid lines), inhibits Trx1 activity in a concentration-dependent manner. (E) HUVEC were incubated with F(ab)2 and NHS for 1 h at 37˚C and C3b deposition was assessed by flow cytometry. (F) Preincubation of HUVEC with blocking Trx-specific F (ab)2 caused an increase in zymosan-mediated MAC deposition. (A)–(D) are each representative of three independently performed experiments. Data in (E) and (F) are shown as means of three independent experiments performed in duplicates with bars indicating SD. Statistical significance of differences was calculated using one-way ANOVA with Bonferroni post- test with *p , 0.05, **p , 0.01, ***p , 0.005. (B and C) Numbers in- dicate molecular mass markers in kilodaltons.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Cytometry, Negative Control, Staining, Western Blot, Incubation, Membrane, Activity Assay, Concentration Assay, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 2. Trx1 inhibits complement-mediated hemolysis in an active site-dependent manner. (A) Reducing 15% SDS-PAGE of purified His- tagged Trx1 (wild-type [WT], active site mutants, and Trx80), followed by silver staining. Lower panel shows schematic of Trx1 protein with muta- tion sites. Δ1: CC/SS variant, C32 and C35 mutated to S; Δ2: Trx80 variant, stop codon inserted after K80; and Δ3: C35S variant, C35 only mutated to S. (B) Functional testing of purified Trx proteins by an insulin reduction assay. Trx proteins and insulin were mixed in the presence (broken lines) or absence (solid lines) of DTT and aggregation of the reduced insulin b-chain followed by absorbance at 650 nm. (C) Sheep RBCs opsonized with Ab were incubated with NHS in DGVB++ buffer, and lysis was measured by the absorbance of released hemoglobin in the supernatant at 490 nm. Trx proteins were added in the presence (broken lines) or absence (solid lines) of 10 mM DTT. (D) The effect of Trx proteins on lysis trig- gered via the alternative activation pathway of complement was studied as in (C), but using rabbit RBCs in Mg++EGTA buffer, which allows the activation of the alternative pathway of complement only. (A) and (B) are representative examples of three independently performed experiments. Results shown in (C) and (D) are the averages of three independent ex- periments performed in duplicate with error bars denoting SD. Statistical analysis was performed by two-way ANOVA. *p , 0.005 as compared with BSA.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: SDS Page, Silver Staining, Variant Assay, Functional Assay, Insulin Reduction Assay, Incubation, Lysis, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 3. Trx1 inhibits activation of complement at the level of the C5 convertase. Plates were coated with aggregated human IgG, mannan, or zymosan to trigger the classical, lectin, or alternative pathways, respec- tively. Plates were then incubated with NHS with varying amounts of Trx proteins, in GVB++ buffer for the classical and lectin pathways, and Mg++

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Activation Assay, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 4. Trx80 interacts with initiators and inhibitors of complement. (A) Complement proteins were coated onto 96-well plates at 5 mg/ml and probed with Trx proteins. Binding was detected using polyclonal anti-Trx, which detects both Trx1 and Trx80. *p , 0.05 and ***p , 0.005 by two-way ANOVA, compared with BSA. (B) 125I-labeled Trx proteins were added to plates coated with complement proteins (solid lines) or BSA (broken lines) and washed, and binding was detected by gamma ray emission. Specificity of binding of [125I]Trx80 was determined by the addition of excess, unlabeled Trx80 as competition during the binding incubation (open triangles). (C) The active-site substrates of Trx1 were investigated using the C35S substrate-trapping mutant of Trx1. Only C4BP and factor H were captured from serum by the C35S mutant in comparison with binding by the CC/SS active site knockout mutant. The difference in binding between C35S and CC/SS was used to determine the active-site specific interaction. **p , 0.01 and ***p , 0.005 by two-way ANOVA. All results in this figure show the averages of at least three independent experiments carried out in duplicates; error bars represent SD. fB, Factor B; fH, factor H; fP, properdin.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Binding Assay, Labeling, Incubation, Mutagenesis, Comparison, Knock-Out

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 6. Trx1 and Trx80 have opposing effects on anaphylatoxin production from plasma. C5a presence was assayed by chemotaxis of neutrophils through a 3-mm pore size membrane, which was fixed and stained, and numbers of cells per field of a 3100 light microscope were counted. To assess the C5a receptor dependency of chemotaxis, cells run with samples in parallel were preincubated with 100 nM C5aRA. (A) Plasma diluted to 3% in Mg++

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Clinical Proteomics, Chemotaxis Assay, Pore Size, Membrane, Staining, Light Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 7. Trx1 inhibits immune complex-mediated neutrophil re- cruitment in vivo. The RPA was carried out in BABLB/c mice. Treatment groups were injected i.v. with 200 mg/ml Trx1 before initiation of the RPA. After 6 h, neutrophil chemotaxis to the site of immune complex formation was assessed by flow cytometry. Representative FACS plots are shown. The data are representative of two independent experiments; four mice per group. *p , 0.05 according to t test.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: In Vivo, Injection, Chemotaxis Assay, Cytometry