Fig 1: KLF10 knockdown promotes activation of LX-2 human HSCs via ATF3. LX-2 cells were transfected with siKLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. The chemical chaperone 4-PBA was applied 2 h before TGF-β1 treatment. (A) Cell viability was determined using MTT assays. Data are expressed as means ± SEM. (B) Levels of α-SMA, fibronectin, and Col1α1 were determined by Western blotting. (C) Collagen accumulation was evaluated by Picro-Sirius red staining. Collagen, red. Scale bar = 100 μm. (D) LX-2 cells were transfected with siKLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for the indicated times. The chemical chaperone 4-PBA was applied 2 h before TGF-β1 treatment. The levels of ATF3, p-SMAD2, p-SMAD3, and SMAD2/3 were determined by Western blotting. GAPDH was used as an internal control.
Fig 2: ATF3 is upregulated in the fibrotic liver of HSD-fed KLF10 KO mice and in activated HSCs. (A) Different genes in the livers of WT and KLF10 KO mice fed a CD or HSD. (B) Principal component (PC) analysis. (C) The 15 most significant GO-BP pathways of the differentially expressed genes between HSD-fed WT and KLF10 KO mice. GO-BP, Gene ontology-biological process. (D) Protein–protein interactions (PPI) generated by Cytoscape. (E) Heatmap of the top 24 upregulated genes between HSD-fed KLF10 KO and WT mice. ATF3 in the pathway of high GO profile and PPI analysis is marked in red. *, ATF3 as a potential target gene of KLF10 during liver fibrosis. The relative mRNA levels of KLF10 (F) and ATF3 (G) were determined by qPCR. LX-2 cells were treated with TGF-β1 for the indicated times. The target gene expression was normalized to the expression of cyclophilin and expressed as means ± SEM. (H) Western blot applied to LX-2 cells treated with TGF-β1 (10 ng/mL) for the indicated times. The levels of α-SMA, fibronectin, Col1α1, KLF10, and ATF3 were determined. GAPDH was used as an internal control. The protein band of Western blotting was quantified by ImageJ and normalized using GAPDH.
Fig 3: KLF10 inhibits TGF-β1-induced activation of LX-2 cells. LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. (A) Cell viability was determined by MTT assays. (B) Protein levels of α-SMA, fibronectin, and Col1α1 were determined by Western blotting. (C) Collagen accumulation was evaluated using Picro-Sirius red staining. Collagen, red. Scale bar = 100 μm. (D) LX-2 cells were transfected with pcDNA-KLF10 for 48 h and treated with TGF-β1 for the indicated times. The levels of p-SMAD2, p-SMAD3, SMAD2/3, KLF10, and ATF3 were determined using Western blotting. β-actin was used as an internal control. (E) LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for another 4 h. mRNA levels of ATF3 were determined by qPCR. Data are expressed as means ± SEM. (F) LX-2 cells were transfected with pcDNA-KLF10 for 48 h and further treated with 10 ng/mL TGF-β1 for 24 h. ATF3 promoter activity was determined by a luciferase assay. * p < 0.05.
Fig 4: KLF10 knockdown promotes TGF-β-induced activation in human LX-2 HSCs. LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and further treated with 10 ng/mL TGF-β1 for another 24 h. (A) Cell viability was determined using MTT assays. (B) Protein levels of α-SMA, fibronectin, and Col1α1 were determined by Western blotting. (C) Collagen accumulation was evaluated by Picro-Sirius red staining. Collagen, red. Scale bars = 100 μm. (D) LX-2 cells transfected with siKLF10 were treated with TGF-β1 for the indicated time periods. The levels of p-SMAD2, p-SMAD3, SMAD2/3, KLF10, and ATF3 were determined by Western blotting. β-actin was used as an internal control. The protein band of Western blotting was quantified by ImageJ and normalized using β-actin. (E) LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and further treated with 10 ng/mL TGF-β1 for another 4 h. The mRNA levels of ATF3 were determined by qPCR. Data are expressed as means ± SEM. (F) LX-2 cells were transfected with siKLF10 (100 pmol) for 48 h and then treated with 10 ng/mL TGF-β1 for 24 h. ATF3 promoter activity was determined using a luciferase assay. * p < 0.05.
Supplier Page from Sino Biological, Inc. for Human KLF10 Gene ORF cDNA clone expression plasmid, N-Flag tag