Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: F12 medium sensitizes lung cancer cells to iron-dependent lipid peroxidation . ( A, B ) Quantification by flow cytometry of oxidized BODIPY-C11 fluorescence in A549 (A) or H838 (B) cells that were cultured in RPMI or F12 medium and treated with 100 μM BSO or vehicle for 24 h. ( C ) Dose response curves for F12-cultured A549 and H838 cells treated with BSO in combination with 5 μM liproxstatin-1 (LIP-1), 5 μM ferrostatin-1 (FER-1) or 50 μM α-tocopherol (α-toco) for 72 h. ( D ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM deferoxamine (DFO) for A549 cells and 9 μM for H838 cells for 72 h. ( E ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM necrostatin-1 (NEC-1) or 10 μM ZVAD-FMK (ZVAD) for 72 h. ( F ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 4 μg/mL certolizumab (CER), 15 μM CU-CPT4a (C4a), 3 μM resatorvid (RST), or 50 μM necrostatin-1 (NEC-1) for 72 h (ND, not done). Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗∗∗P < 0.0001.

Article Snippet: Drugs used were auranofin (A6733; Sigma-Aldrich), α-tocopherol (T3251; Sigma-Aldrich), bafilomycin A1 (SML1661; Sigma-Aldrich), certolizumab pegol (HYP9953; MedChemExpress), chloroquine (C6628; Sigma-Aldrich), CU-CPT4A (HY-108473; MedChemExpress), deferoxamine (D9533; Sigma-Aldrich), erastin (e7781; Sigma Aldrich), FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) (HY-100410; MedChemExpress), ferrostatin-1 (SML0583; Sigma-Aldrich), l -buthionine-sulfoximine (b2515; Sigma-Aldrich), mito-TEMPO (HY–W001187; MedChemExpress), necrostatin-1 (N9037; Sigma-Aldrich), liproxstatin-1 (SML1414; Sigma-Aldrich), oligomycin (HY–N6782; MedChemExpress), puromycin dihydrochloride (A1113803; Gibco), rotenone (HY–B1756; MedChemExpress), RSL3 (SML2234; Sigma-Aldrich), resatorvid (HY-11109; MedChemExpress), and Z-VAD-FMK (V116; Sigma-Aldrich).

Techniques: Flow Cytometry, Fluorescence, Cell Culture

Journal: Pharmaceuticals

Article Title: Aronia Berry Extract Inhibits Cancer Stemness and Overcomes 5-Fluorouracil Resistance by Targeting TLR3/NF-κB Signaling in Colorectal Cancer

doi: 10.3390/ph19020261

Figure Lengend Snippet: The expression of TLR3 is positively correlated with 5-FU-Resistance in CRC cells. ( A ) Schematic overview of the transcriptomic analysis workflow comparing parental and 5-FU-R HCT116 and SW480 CRC cell lines using RNA-sequencing data ( GSE196900 ). Differentially upregulated genes were identified using the criteria of log 2 fold change (log 2 FC) > 1.5 and p < 0.05, and overlapping upregulated genes between the two 5-FU-R cell lines were determined. ( B ) Scatter plot of KEGG pathway enrichment analysis based on genes upregulated in 5-FU-R CRC cells compared with their parental counterparts. The size of each circle represents the number of differentially expressed genes in the corresponding pathway, while the color scale indicates the enrichment p -value. ( C ) qRT-PCR analysis showing increased relative TLR3 mRNA expression in 5-FU-R HCT116 cells compared with parental HCT116 cells. ( D ) qRT-PCR analysis of TLR3 mRNA expression in 5-FU-R SW480 cells relative to parental SW480 cells. Gene expression levels were normalized to GAPDH and expressed relative to parental control. ( E ) Representative phase-contrast images showing morphological changes and enhanced formation of adherent spheroid-like clusters in 5-FU-R CRC cells following stimulation with the TLR3 agonist poly (I:C) (1 μg/mL) for the indicated time points. ( F ) Cell viability analysis of 5-FU-R HCT116 cells treated with 5-FU in the presence of DMSO or the TLR3 inhibitor CU-CPT 4a, as determined by the MTT assay. ( G ) Cell viability analysis of 5-FU-R SW480 cells treated with 5-FU following inhibition of TLR3 signaling using CU-CPT 4a. Data are presented as mean ± SD. * p < 0.05 vs. parental cells or DMSO-treated control, as indicated.

Article Snippet: To inhibit TLR3 signaling, cells were treated with the TLR3 inhibitor CU-CPT 4a (MCE).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Gene Expression, Control, MTT Assay, Inhibition

Journal: Pharmaceuticals

Article Title: Aronia Berry Extract Inhibits Cancer Stemness and Overcomes 5-Fluorouracil Resistance by Targeting TLR3/NF-κB Signaling in Colorectal Cancer

doi: 10.3390/ph19020261

Figure Lengend Snippet: The combination treatment of 5-FU and ABE has been shown to regulate the TLR3/NF-κB axis in 5-FU-R CRC cell lines. ( A ) Western blot analysis of TLR3 and NF-κB protein expression in 5-FU-R HCT116 and SW480 cells treated with 5-FU, ABE, or their combination for 48 h. Combined treatment resulted in a marked reduction in TLR3 and NF-κB protein levels compared with untreated cells or either monotherapy. Representative immunoblots and corresponding densitometric quantification normalized to GAPDH are shown. ( B ) Western blot analysis of NF-κB expression in cytoplasmic and nuclear fractions of 5-FU-R CRC cells following treatment with 5-FU, ABE, or their combination for 48 h. Combined treatment significantly reduced NF-κB levels in both the cytoplasm and the nucleus, indicating inhibition of NF-κB activation and nuclear translocation. β-actin was used as a loading control for cytoplasmic fractions, and Lamin A/C was used as a nuclear loading control. Quantitative densitometric analyses are shown below each blot. Data are presented as mean ± SD. * p indicates p < 0.05 vs. control group; # p indicates p < 0.05 vs. combination group.

Article Snippet: To inhibit TLR3 signaling, cells were treated with the TLR3 inhibitor CU-CPT 4a (MCE).

Techniques: Western Blot, Expressing, Inhibition, Activation Assay, Translocation Assay, Control

Journal: Pharmaceuticals

Article Title: Aronia Berry Extract Inhibits Cancer Stemness and Overcomes 5-Fluorouracil Resistance by Targeting TLR3/NF-κB Signaling in Colorectal Cancer

doi: 10.3390/ph19020261

Figure Lengend Snippet: The combination of 5-FU and ABE effectively exhibits anti-cancer activity in CRC patient-derived organoid models. ( A ) Representative bright-field images of colorectal cancer PDOs treated with 5-FU, ABE, or their combination for 48 h. Combined treatment markedly reduced organoid size and overall growth compared with untreated controls or either monotherapy. ( B ) qRT-PCR analysis of TLR3 mRNA expression in CRC PDOs following the indicated treatments. Transcript levels were normalized to GAPDH and expressed relative to the untreated control. ( C ) Representative confocal immunofluorescence images showing NF-κB expression in CRC organoids treated with DMSO, 5-FU, ABE, or the combination for 48 h. Reduced NF-κB staining was observed in organoids treated with the combination therapy compared with single-agent treatments. * p indicates p < 0.05 vs. control group; # p indicates p < 0.05 vs. combination group.

Article Snippet: To inhibit TLR3 signaling, cells were treated with the TLR3 inhibitor CU-CPT 4a (MCE).

Techniques: Activity Assay, Derivative Assay, Quantitative RT-PCR, Expressing, Control, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Differentially expressed genes (DEGs) in full-thickness bladder tissues from HIC patients and controls identified via RNA sequencing (|log₂FC|≥2; adjusted p < 0.05, two-sided Wald test using DESeq2). Gene Ontology (GO) ( B ) and KEGG ( C ) enrichment analyses of DEGs (two-sided hypergeometric test with FDR correction). D UMAP visualization of single-cell transcriptomic landscape of urothelium, with urothelial cells (UCs) identified by KRT19 and UPK1A expression. GO ( E ) and KEGG ( F ) analyses of upregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). GO ( G ) and KEGG ( H ) analyses of downregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). I , J Gene Set Enrichment Analysis (GSEA) of altered pathways in UCs (two-sided permutation test with 1000 permutations, with multiple testing corrected using the FDR). The exact p value for TLR signaling of UCs in panel I is 0.0009. K Expression of Toll-like receptor (TLR) subtypes in UCs from HIC bladders. mRNA ( L ) and protein ( M ) expression levels of TLR3 in isolated urothelium from control and HIC patients ( n = 7 control vs. 10 HIC; data show median (IQR); two-sided Mann–Whitney U test; ns not significant; Bar for panel M: 1 cm). N Immunostaining showing the distribution of TLR3 protein in the urothelium of patients with HIC ( n = 7 control vs. 10 HIC; one section and field per patient; Bar: 100 μm). IHC immunohistochemistry, HL Hunner lesions, U urothelium, LP lamina propria (blue line), M muscularis. Source data are provided as a Source Data file.

Article Snippet: TLR3 inhibitor group: Same intravesical TCDCA instillation as above, followed by intraperitoneal injection of a TLR3 inhibitor (CU-CPT 4a, MCE, Cat. HY-108473) at 1 mg/kg , twice per week for 2 weeks.

Techniques: RNA Sequencing, Expressing, Isolation, Control, MANN-WHITNEY, Immunostaining, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Experimental workflow of E. avium colonization in antibiotic-pretreated mice (Abx-mice, n = 6 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . B Increased abundance of E. avium confirmed by metagenomic sequencing ( n = 6 per group). C Evaluation of voiding behavior following E. avium transplantation ( n = 6 per group; one measurement per mouse). D Measurement of mechanical pain threshold post-transplantation ( n = 6 per group). E Representative histological analysis of bladder tissues after E. avium transplantation ( n = 6 per group; one section and field per mouse). F , G Quantification of blood and urinary bile acids using comprehensive targeted bile acid profiling ( n = 6 per group). H Cell viability of human urothelial cells (HUCs) following TCDCA or TUDCA treatment ( n = 3 independent experiments). I Expression of ZO-1, TNF-α, and TLR3 following TCDCA (200 µM) exposure ( n = 3 independent experiments). J Experimental workflow of intravesical instillation of TCDCA in rats ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . K Evaluation of voiding function after TCDCA instillation ( n = 5 per group; each rate measured once). L Assessment of mechanical pain threshold following TCDCA exposure ( n = 5 per group). M Representative histology of bladder tissues post-instillation ( n = 5 per group; one section per rat, one field quantified per section). For panels ( B – H , K , L ): data are presented as median (IQR). Statistical analysis was performed using the two-sided Mann–Whitney U test. IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: TLR3 inhibitor group: Same intravesical TCDCA instillation as above, followed by intraperitoneal injection of a TLR3 inhibitor (CU-CPT 4a, MCE, Cat. HY-108473) at 1 mg/kg , twice per week for 2 weeks.

Techniques: Sequencing, Transplantation Assay, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A , B Expression of tight junction protein ZO-1 and inflammatory marker TNF-α after TLR3 intervention in TCDCA-pretreated human urothelial cells (HUCs) ( n = 3 independent experiments). C Transepithelial resistance (TER) changes following TLR3 intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). D , E Expression of ZO-1 and TNF-α after pentosan polysulfate sodium (PPS) administration in TCDCA-pretreated HUCs ( n = 3 independent experiments). F TER changes following PPS intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). G Experimental workflow showing TLR3 inhibitor and PPS administration to rats following intravesical TCDCA instillation ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . H Assessment of voiding function (one measurement per rat) and pain threshold after TLR3 inhibition ( n = 5 per group). I Representative histological images of bladder tissues post-TLR3 inhibition ( n = 5 per group; one section and field per rat). For panels ( B , C , E , F , H ): data are presented as median (IQR). Statistical analysis was performed using the Kruskal–Wallis test. ns not significant, IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: TLR3 inhibitor group: Same intravesical TCDCA instillation as above, followed by intraperitoneal injection of a TLR3 inhibitor (CU-CPT 4a, MCE, Cat. HY-108473) at 1 mg/kg , twice per week for 2 weeks.

Techniques: Expressing, Marker, Inhibition, Immunohistochemistry