tlr3 inhibitor cu cpt 4a  (MedChemExpress)

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

Journal: Life Science Alliance

Article Title: TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes

doi: 10.26508/lsa.202402777

Figure Lengend Snippet: (A) Representative images of n ormal h uman e pidermal k eratinocytes 72 h after UVB (10 mJ/cm 2 ) exposure or poly(I:C) (20 μg/ml) treatment. Cells exposed to UVB are given fresh media 24 h after exposure and left in culture for another 48 h; poly(I:C) is on the cells during the first 24 h, followed by a washout period of 48 h (72 h in total). Images were captured at 10X magnification; scale bar represents 100 μm. (B) Quantification of spindle-like cells 48 and 72 h after UVB (10 mJ/cm 2 ) exposure, n ≥ 21 images quantified. (C) Quantification of spindle-like cells 48 and 72 h after poly(I:C) (20 μg/ml), n ≥ 24 images quantified. (D) IL-6 ELISA data from the media of keratinocytes exposed to UVB (10 mJ/cm 2 ) or treated with poly(I:C) (20 μg/ml) 24 and 48 h after treatment, unpaired t test, two-tailed, P < 0.05, n ≥ 4. (E) TLR3 gene expression by qRT–PCR 48 h after UVB (10 mJ/cm 2 ). Data were normalized to RPLP0 , n ≥ 16. (F) TLR3 gene expression by qRT–PCR 48 h after poly(I:C) (20 μg/ml). Data were normalized to RPLP0 , n ≥ 36. (G) Representative immunoblot from keratinocytes exposed to UVB (10 mJ/cm 2 ) and poly(I:C) (20 μg/ml) shows increases in TLR3 protein (full-length and cleaved) by immunoblot, n = 3. (H) Number of differentially expressed genes in 72-h UVB-treated cells and poly(I:C)-treated cells and their overlap. The number of overlapping genes is significant using Fisher’s exact test, P = 0. (I) Hallmark gene sets of interest. NES, normalized enrichment score. (J, K) Volcano plots illustrating changes in the genes involved in epithelial-to-mesenchymal transition after (J) UVB and (K) poly(I:C) treatment. * denotes significance compared with the control, P < 0.05, Mann–Whitney t test, two-tailed, unless otherwise noted above.

Article Snippet: TLR3 pharmacological inhibitor (CU CPT 4a; (R,Z)-2-(((3-chloro-6-fluorobenzo[b]thiophen-2-yl)(hydroxy)methylene)amino)-3-phenylpropanoic acid) was obtained from Tocris Biologicals, and a stock solution of 100 mM was made in DMSO and stored at −20C.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Quantitative RT-PCR, Western Blot, Control, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes

doi: 10.26508/lsa.202402777

Figure Lengend Snippet: (A) TLR3 gene expression of NHEKs pre-treated with competitive TLR3 inhibitor CU CPT 4A (80 μM) for 1 h before UVB (10 mJ/cm 2 ) exposure. Cells were collected 72 h after UVB. Data were normalized to RPLP0. * denotes significance compared with the control, # significance compared with UVB, Mann–Whitney t test, two-tailed, P < 0.05, n = 6. (B) TLR3 gene expression of NHEKs pre-treated with CU CPT 4A (80 μM) for 1 h before poly(I:C) (20 μg/ml) treatment. Cells were harvested 24 h after poly(I:C). Data were normalized to RPLP0 , Mann–Whitney t test, two-tailed. * denotes significance compared with the control, # significance compared with poly(I:C), P < 0.05, n ≥ 10. (C) Chemical inhibition of TLR3 negates TLR3 protein expression in keratinocytes, representative image, n = 4. (D) Morphology of keratinocytes exposed to UVB (10 mJ/cm 2 ) and CU CPT 4a (80 μM) 72 h after exposure, scale bar = 100 μm. (E) Morphology of keratinocytes treated with poly(I:C) (20 μg/ml) and CU CPT 4a (80 μM) 72 h after exposure, scale bar = 100 μm. (F) Chemical inhibition of TLR3 activation before UVB exposure attenuates epithelial-to-mesenchymal transition–associated marker gene expression 72 h after exposure. Data were normalized to RPLP0 . * denotes significance compared with the control, # significance compared with UVB, Mann–Whitney t test, one-tailed, P < 0.05, n = 6. (G) Chemical inhibition of TLR3 activation before poly(I:C) treatment attenuates epithelial-to-mesenchymal transition–associated marker gene expression 72 h after treatment, normalized to RPLP0 . * denotes significance compared with the control, # significance compared with poly(I:C), Mann–Whitney t test, one-tailed, P < 0.05, n ≥ 14.

Article Snippet: TLR3 pharmacological inhibitor (CU CPT 4a; (R,Z)-2-(((3-chloro-6-fluorobenzo[b]thiophen-2-yl)(hydroxy)methylene)amino)-3-phenylpropanoic acid) was obtained from Tocris Biologicals, and a stock solution of 100 mM was made in DMSO and stored at −20C.

Techniques: Gene Expression, Control, MANN-WHITNEY, Two Tailed Test, Inhibition, Expressing, Activation Assay, Marker, One-tailed Test

Journal: Life Science Alliance

Article Title: TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes

doi: 10.26508/lsa.202402777

Figure Lengend Snippet: (A) Treatment with chemical inhibitors limits EMT-associated protein changes induced by UVB treatment at 48 and 72 h, representative image. (B) Protein level was semi-quantified by densitometric analysis. Samples were normalized to GAPDH; fold change was calculated compared with the control, n ≥ 3. (C) Treatment with chemical inhibitors limits EMT-associated protein changes induced by poly(I:C) treatment at 48 and 72 h, representative image. (D) Protein level was semi-quantified by densitometric analysis. Samples were normalized to GAPDH; fold change was calculated compared with the control, n ≥ 3. (E) TLR3 KD CRISPR keratinocytes have decreased EMT-associated protein changes induced by UVB treatment at 48 and 72 h compared with normal controls, representative image. (F) Relative density of fibronectin, vimentin, and E-cadherin was normalized to GAPDH. Fold change was calculated compared with the control, n = 3. (G) TLR3 KD CRISPR keratinocytes have decreased EMT-associated protein changes induced by poly(I:C) at 48 and 72 h compared with normal controls, representative image. (H) Relative density of fibronectin, vimentin, and E-cadherin was normalized to GAPDH. Fold change was calculated compared with the control, n = 3. * denotes significance compared with the control, # significance compared with UVB or poly(I:C), paired t test, one-tailed, P < 0.05.

Article Snippet: TLR3 pharmacological inhibitor (CU CPT 4a; (R,Z)-2-(((3-chloro-6-fluorobenzo[b]thiophen-2-yl)(hydroxy)methylene)amino)-3-phenylpropanoic acid) was obtained from Tocris Biologicals, and a stock solution of 100 mM was made in DMSO and stored at −20C.

Techniques: Control, CRISPR, One-tailed Test

Journal: Life Science Alliance

Article Title: TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes

doi: 10.26508/lsa.202402777

Figure Lengend Snippet: (A) Schematic showing the CRISPR gDNA cut site upstream of the essential TLR3 ubiquitination site (K831) required for TLR3 transport from ER to endolysosomes where it is activated by proteolytic cleavage . (B) TLR3 CRISPR/Cas9 cells have less activated cleaved TLR3 protein expression than normal h uman e pidermal k eratinocytes 24 h after poly(I:C) (20 μg/ml) stimulation. TLR3 CRISPR B, C, and D represent three independent pools of primary KD keratinocytes. (C) Images of TLR3 CRISPR keratinocytes, representative of three independent experiments; the scale bar represents 100 μm.

Article Snippet: TLR3 pharmacological inhibitor (CU CPT 4a; (R,Z)-2-(((3-chloro-6-fluorobenzo[b]thiophen-2-yl)(hydroxy)methylene)amino)-3-phenylpropanoic acid) was obtained from Tocris Biologicals, and a stock solution of 100 mM was made in DMSO and stored at −20C.

Techniques: CRISPR, Ubiquitin Proteomics, Expressing

Journal: Life Science Alliance

Article Title: TLR3 activation mediates partial epithelial-to-mesenchymal transition in human keratinocytes

doi: 10.26508/lsa.202402777

Figure Lengend Snippet: (A) Gene expression of TLR3 and non-melanoma skin cancer (NMSC)–associated genes in clinically normal skin from both unexposed and sun-exposed body areas, qRT–PCR, normalized to RPLP0 , n ≥ 4 patient samples per group. (B) Gene expression of NMSC-associated genes in n ormal h uman e pidermal k eratinocytes treated with TLR3 agonist poly(I:C) (20 μg/ml) for 24 h followed by a 24-h washout. Data were normalized to RPLP0 , n ≥ 11. (C) Gene expression of TLR3 - and NMSC-associated genes in n ormal h uman e pidermal k eratinocytes pre-treated with CU CPT 4A (80 μM) for 1 h before poly(I:C) (20 μg/ml) treatment. Cells were harvested 48 h after poly(I:C). Data were normalized to RPLP0 , # significance compared with poly(I:C), P < 0.05, n ≥ 20. (D) TLR3 protein expression in well- and moderately differentiated cutaneous squamous cell carcinomas. Tumors were graded by H&E and stained for TLR3 and IgG by IHC (n = 13 individual tumors), images were captured at 20X, and the scale bar represents 50 μm. (E) TLR3 protein expression in graded basal cell carcinomas. Tumors were graded by H&E and stained for TLR3 and IgG by IHC (n = 8 individual tumors), images were captured at 20X magnification, and the scale bar represents 50 μm. * denotes significance compared with the control, P < 0.05, Mann–Whitney t test, two-tailed.

Article Snippet: TLR3 pharmacological inhibitor (CU CPT 4a; (R,Z)-2-(((3-chloro-6-fluorobenzo[b]thiophen-2-yl)(hydroxy)methylene)amino)-3-phenylpropanoic acid) was obtained from Tocris Biologicals, and a stock solution of 100 mM was made in DMSO and stored at −20C.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Staining, Control, MANN-WHITNEY, Two Tailed Test