tgf β 1  (R&D Systems)

Journal: Advanced Science

Article Title: Nuclear Factor I‐B Delays Liver Fibrosis by Inhibiting Chemokine Ligand 5 Transcription

doi: 10.1002/advs.202511311

Figure Lengend Snippet: NFIB transcriptionally inhibits CCL5 induction in HSCs. (A) A CCL5 promoter luciferase (−1000) was transfected to LX‐2 cells with increasing doses of NFIB. Luciferase activities were normalized by protein concentration and green fluorescent protein. N = 3. (B) Hypergeometric optimization of motif enrichment analysis. (C) Truncated CCL5 promoter–luciferase constructs were transfected into LX‐2 cells with or without NFIB. Luciferase activities were normalized by protein concentration and green fluorescent protein. N = 3. (D) LX‐2 cells were treated with TGF‐β (2 ng/mL) and harvested at the indicated time points. ChIP assays were performed with anti‐NFIB or IgG. N = 3. (E) Re‐ChIP assays were performed with the indicated antibodies. N = 3. (F) Wild‐type and mutated CCL5 promoter‐luciferase constructs were transfected into HEK293 cells with or without NFIB. Luciferase activities were normalized by protein concentration and green fluorescent protein. N = 3. (G) The mRNA and protein levels of CCL5 were detected by PCR and ELISA. N = 3. LX‐2 cells were treated with recombinant CCL5 (20 ng/mL) for 24 h after NFIB overexpression. (H) The mRNA levels of Acta2, Postn , and NFIB in LX‐2 cells were measured by quantitative PCR. N = 3. (I) Boyden chamber transwell assay (100×). N = 3. (J) Cell proliferation was evaluated using EdU (200×). N = 3. (K) Collagen contraction assay. N = 3. Data are expressed as the mean±SD. * p < 0.05, two‐tailed Student's t ‐test.

Article Snippet: Prior to TGF‐β (2 ng/mL; #246‐LP‐025; R&D System, Minneapolis, MN, USA) stimulation, LX‐2 cells were subjected to an overnight serum starvation, which imitated HSC activation in vitro [ ].

Techniques: Luciferase, Transfection, Protein Concentration, Construct, Enzyme-linked Immunosorbent Assay, Recombinant, Over Expression, Real-time Polymerase Chain Reaction, Boyden Chamber Transwell Assay, Contraction Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Nuclear Factor I‐B Delays Liver Fibrosis by Inhibiting Chemokine Ligand 5 Transcription

doi: 10.1002/advs.202511311

Figure Lengend Snippet: Juglone attenuates HSC activation and MASH fibrosis. (A‐C) LX‐2 cells were subjected to TGF‐β1 (2 ng/mL) treatment for 24 h with or without juglone treatments (3, 10, or 30 µ m ). Then, all cells were harvested for studies as follows. (A) The mRNA levels of Acta2 and NFIB in LX‐2 cells were measured by qPCR. N = 3. (B) Collagen contraction assay. N = 3. (C) Boyden chamber transwell assay (100×). N = 3. (D–H) C57BL/6 mice were fed on an CDAHFD diet for 8 weeks, during which they concurrently received different doses of Juglone (0.25 and 1.0 mg/kg) via oral gavage. Hepatic profibrogenic genes in mouse liver tissues were examined using quantitative PCR (D) and Western blotting (E). N = 3–7. (F) Liver sections were stained with H&E (50×), picrosirius red (50×), Masson's (50×) and IHC (100×) (CD3, F4/80) staining. N = 7. (G) IF staining (50×) for 4‐HNE expression in liver tissues. N = 7. (H) Plasma AST and ALT levels. N = 7. (I) Hepatic hydroxyproline levels. N = 7. Data are expressed as the mean±SD. * p < 0.05, two‐tailed Student's t ‐test.

Article Snippet: Prior to TGF‐β (2 ng/mL; #246‐LP‐025; R&D System, Minneapolis, MN, USA) stimulation, LX‐2 cells were subjected to an overnight serum starvation, which imitated HSC activation in vitro [ ].

Techniques: Activation Assay, Contraction Assay, Boyden Chamber Transwell Assay, Real-time Polymerase Chain Reaction, Western Blot, Staining, Expressing, Clinical Proteomics, Two Tailed Test