Journal: Immune Network

Article Title: TXNIP Suppresses Lung Cancer Progression by Inhibiting TRAF6-Mediated NF-κB Activation and Autophagy

doi: 10.4110/in.2025.25.e33

Figure Lengend Snippet: (A) HEK293T cells were transfected with Mock, HA-Ub, Flag-TRAF6, or different concentrations of Myc-TXNIP, as indicated. At 24 h after transfection, transfected cells were used for protein extraction. Proteins were immunoprecipitated with an anti-Flag Ab. IB assay was then performed with anti-Flag, anti-Myc, or anti-HA Ab. (B) HEK293T cells were transfected with Mock, Flag-TRAF6, HA-Ub, different concentrations of Flag-TXNIP, or Myc-TAK1, as indicated. At 24 h after transfection, transfected cells were used for protein extraction. Proteins were then immunoprecipitated with anti-Myc Ab. IB assay was performed with anti-Myc, anti-Flag, anti-TRAF6, or anti-HA Ab. (C) 293/hTLR4A cells were transfected with Mock or different concentrations of Flag-TXNIP, along with the pBIIx-luc NF-κB-dependent reporter construct and the Renilla luciferase vector. At 24 h post transfection, cells were treated or not with LPS (500 ng/ml) for 6 h and lysed. Luciferase activity was measured using a dual luciferase assay kit. Results are presented as ± SD in three independent experiments. (D) A schematic model showing how TXNIP inhibits NF-κB activation through the TRAF6-TAK1 signaling axis. * p<0.05, ** p<0.01, and *** p<0.001.

Article Snippet: TXNIP human Tagged ORF clone (#RG210804) and TAK1 (MAP3K7) (#RG213823) human Tagged ORF clone were purchased from Origene (Rockville, MD, USA).

Techniques: Transfection, Protein Extraction, Immunoprecipitation, Construct, Luciferase, Plasmid Preparation, Activity Assay, Activation Assay

Journal: Immune Network

Article Title: TXNIP Suppresses Lung Cancer Progression by Inhibiting TRAF6-Mediated NF-κB Activation and Autophagy

doi: 10.4110/in.2025.25.e33

Figure Lengend Snippet: (A, B) Ctrl H1299 and TXNIP -KO H1299 cells were treated with vehicle (DMSO, 0.1% v/v concentration), LPS (15 µg/ml), or Poly(I:C) (20 µg/ml) for different time periods (A). The residual gap between migrating cells from the opposing wound edge is expressed as a percentage of the initial scraped area (± SD, n=3 different plates) (B). (C, D) Ctrl H1299 and TXNIP -KO H1299 cells were treated with vehicle (DMSO, 0.1% v/v concentration), LPS (15 µg/ml), or Poly(I:C) (15 µg/ml) (C). The number of migrating cells was counted. Results are presented as mean ± SD of 3 independent experiments (D). (E, F) Ctrl H1299 and TXNIP -KO H1299 cells were treated with vehicle (DMSO, 0.1% v/v concentration), LPS (15 µg/ml), or Poly(I:C) (10 µg/ml). Anchorage-independent colony formation assay was performed (E), as described in Materials and Methods. The number of colonies was measured using Adobe Photoshop software. Results are presented as mean ± SD of 3 independent experiments (F, n=3 plates). (G, H) Ctrl H1299 and TXNIP -KO H1299 cells were treated with vehicle (DMSO, 0.1% v/v concentration), LPS (5 µg/ml, G), or Poly(I:C) (4 µg/ml, H). MTT assay was then performed as described in in Material and Methods. Results are presented as mean ± SD of 3 independent experiments. (I, J) 3D spheroid formation assays were performed using Ctrl A549 and TXNIP -KO A549 cells, which were seeded in 96-well plates and incubated at 37°C for 48 h to allow spheroid formation. Spheroids were then treated with either LPS (3 µg/ml) or Poly(I:C) (1 µg/ml) and cultured further as indicated. Spheroid size was measured using ImageJ software, and images were captured using phase-contrast microscopy (I, scale bar=100 µm). Data are presented as mean ± SD (J, n=5). Statistical significance (Student’s t -test). (K) A schematic model illustrating how TXNIP inhibits lung cancer progression: Upon TLR3 or TLR4 stimulation, TXNIP binds to TRAF6, preventing its dimerization and auto-ubiquitination. This inhibition suppresses both the TRAF6-BECN1 axis for autophagy and the TRAF6-TAK1 signaling axis for NF-κB activation, ultimately reducing lung cancer progression. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. # p<0.05 and ## p<0.01 for comparisons between TXNIP -KO A549 and Ctrl A549 cells (in ).

Article Snippet: TXNIP human Tagged ORF clone (#RG210804) and TAK1 (MAP3K7) (#RG213823) human Tagged ORF clone were purchased from Origene (Rockville, MD, USA).

Techniques: Concentration Assay, Colony Assay, Software, MTT Assay, Incubation, Cell Culture, Microscopy, Ubiquitin Proteomics, Inhibition, Activation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of Fpr2 expression protects against endotoxin-induced acute lung injury by interacting with Nrf2-regulated TAK1 activation.

doi: 10.1016/j.biopha.2020.109943

Figure Lengend Snippet: Fig. 5. Fpr2-mediated ALI requires activation of TAK1 signaling. (A) RAW264.7 cells were transfected with varying amounts of Flag-tagged Fpr2 for 24 h, and then were incubated in the absence or presence of LPS (100 ng/ml) for another 24 h. Then, all cells were harvested for western blot analysis of p-TAK1 and Flag. (B) RAW264.7 cells were transfected with siTAK1 or oeTAK1 for 24 h, followed by transfection efficacy analysis using western blotting. (C,D) RAW264.7 cells were transfected with siFpr2 combined with or without oeTAK1 for 24 h, and then were subjected to LPS (100 ng/ml) for another 24 h. (C) RT-qPCR analysis was used to calculate TNF-α, IL-1β and IL-6 mRNA expression levels in cells. (D) Western blot analysis was performed to determine p-NF-κB, p-p38, p-ERK1/2 and p-JNK protein expression levels in cells. (E–G) RAW264.7 cells were transfected with oeFpr2 combined with or without siTAK1 for 24 h, followed by LPS (100 ng/ml) incubation for another 24 h. (E) TNF-α, IL-1β and IL-6 mRNA levels were measured by RT-qPCR analysis. (F,G) The protein expression levels of p-NF-κB, p-p38, p-ERK1/2 and p- JNK in cells were assessed using western blot analysis. (H) A model demonstrating how Fpr2 signaling regulated LPS-induced ALI. Data represented as mean ± SEM. #P < 0.05 and ##P < 0.01.

Article Snippet: For Fpr2 and TAK1 overexpression, the entire open reading frame of Fpr2 and TAK1 cloned in the pCDNA3-vector (oe-NC), pCDNA3-Fpr2 (oeFpr2) and pCDNA3-TAK1 (oeTAK1) were purchased from Origene (Rockville, USA).

Techniques: Activation Assay, Transfection, Incubation, Western Blot, Quantitative RT-PCR, Expressing