Journal: Liver International

Article Title: Curcumin Targets Crispld2 to Suppress Hepatic Stellate Cell Activation via PI3K / AKT Pathway Inhibition in Hepatic Fibrosis

doi: 10.1111/liv.70696

Figure Lengend Snippet: Curcumin suppresses HSC activation by targeting Crispld2. Animal groups: Control, CCl 4 , CCl 4 + Cur (25 mg/kg), CCl 4 + Cur (50 mg/kg); n = 5. (A) QRT‐PCR measuring the mRNA level of Crispld2. (B) WB determining the protein level of Crispld2. Groupings of LX‐2 cells: Oe‐NC, oe‐Crispld2. (C) QRT‐PCR verifying transfection efficiency. (D) WB confirming transfection efficiency. Groupings of LX‐2 cells with curcumin treatment: Control, TGF‐β + DMSO + oe‐NC, TGF‐β + Cur + oe‐NC, and TGF‐β + Cur + oe‐Crispld2. (E) QRT‐PCR detecting the mRNA levels of Crispld2. (F) WB examining the protein levels of Crispld2. (G) CCK‐8 assay assessing cell viability. (H) Flow cytometry measuring apoptosis. (I) WB detecting the expression of fibrotic proteins α‐SMA, collagen I, fibronectin, and TIMP1. (J, K) ELISA measuring the levels of inflammatory cytokines IL‐6 (J) and TNF‐α (K). * p < 0.05.

Article Snippet: We cultured human HSC line LX‐2 (RRID: CVCL_5792; BNCC337957, BNCC, China) in RPMI‐1640 complete medium (BNCC, China) containing 10% FBS (Beyotime, China) and human embryonic kidney 293 T cells (RRID: CVCL_0063; BNCC353535, BNCC, China) in DMEM‐H complete medium (BNCC, China) containing 10% FBS and 2 mM L‐glutamine (MCE, USA).

Techniques: Activation Assay, Control, Quantitative RT-PCR, Transfection, CCK-8 Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Liver International

Article Title: Curcumin Targets Crispld2 to Suppress Hepatic Stellate Cell Activation via PI3K / AKT Pathway Inhibition in Hepatic Fibrosis

doi: 10.1111/liv.70696

Figure Lengend Snippet: Curcumin treatment suppresses HSC activation by modulating the Crispld2 to mediate the PI3K/AKT axis. (A) KEGG enrichment of Crispld2 in the transitional HSC treatment group. Animal groups: Control, CCl 4 , CCl 4 + Cur (25 mg/kg), CCl 4 + Cur (50 mg/kg); n = 5. (B) WB assessing the protein levels of p‐PI3K, PI3K, p‐AKT, and AKT. Groups of LX‐2 cells treated by TGF‐β: Sh‐NC, sh‐Crispld2. (C) QRT‐PCR quantification of Crispld2 mRNA. (D) WB analysis of Crispld2, p‐PI3K, PI3K, p‐AKT, AKT. Experimental groups of LX‐2 cells with Crispld2 overexpression induced by TGF‐β, following curcumin or LY294002 treatment: Control, Cur + oe‐NC, Cur + oe‐Crispld2, Cur + oe‐Crispld2 + LY294002. ( E) Co‐IP assays confirmed the interaction of CRISPLD2 with PI3K and AKT. (F) CCK‐8 assessing cell viability. (G) Flow cytometry assessing apoptosis. (H) WB for p‐PI3K, PI3K, p‐AKT, AKT and fibrotic proteins (α‐SMA, collagen I, fibronectin, TIMP1). (I, J) ELISA detection of inflammatory cytokines IL‐6 (I) and TNF‐α (J). * p < 0.05.

Article Snippet: We cultured human HSC line LX‐2 (RRID: CVCL_5792; BNCC337957, BNCC, China) in RPMI‐1640 complete medium (BNCC, China) containing 10% FBS (Beyotime, China) and human embryonic kidney 293 T cells (RRID: CVCL_0063; BNCC353535, BNCC, China) in DMEM‐H complete medium (BNCC, China) containing 10% FBS and 2 mM L‐glutamine (MCE, USA).

Techniques: Activation Assay, Control, Quantitative RT-PCR, Over Expression, Co-Immunoprecipitation Assay, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: The transcription factor SMC1A promotes LAMC2 expression to induce HSC activation in vitro. (A) The transcription factors regulating LAMC2 in the hTFtarget database were intersected with differentially expressed genes in the two GEO datasets. (B) The binding peaks of SMC1A in the promoter region of LAMC2 were analyzed using the ChIP‐seq database. (C, D) The mRNA expression of SMC1A and LAMC2 in TGF‐β1‐stimulated LX‐2 cells and those preinfected with sh‐NC or sh‐SMC1A was examined via RT‐qPCR ( n = 3). The expression of the profibrotic genes PDGFRA (E), TIMP1 (F), and ACTA2 (G) in LX‐2 cells was examined via RT‐qPCR ( n = 3). The localization (H) and expression (I) of SMC1A in LX‐2 cells were examined via immunofluorescence experiments ( n = 3). (J) The enrichment of SMC1A in the LAMC2 promoter region in LX‐2 cells was examined via ChIP‐PCR; immunoprecipitation was performed using an anti‐SMC1A antibody ( n = 3). (K) Dual‐luciferase assay of the promoter transcriptional activity of LAMC2 in LX‐2 cells in the presence of sh‐SMC1A ( n = 3). Representative immunofluorescence images and quantification of collagen I (L, M) and α‐SMA (N, O) in LX‐2 cells ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. An unpaired t ‐test or one‐way ANOVA was used for statistical analysis. ACTA2, actin alpha 2, smooth muscle; ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; HSC, hepatic stellate cell; LAMC2, laminin subunit gamma 2; PDGFRA, platelet‐derived growth factor receptor alpha; RT‐qPCR, reverse transcription quantitative PCR; SMC1A, structural maintenance of chromosome protein 1A; TIMP1, TIMP metallopeptidase inhibitor 1.

Article Snippet: HSC LX‐2 cells (CL‐0560) were procured from Procell.

Techniques: Expressing, Activation Assay, In Vitro, Binding Assay, ChIP-sequencing, Quantitative RT-PCR, Immunofluorescence, Immunoprecipitation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Cell Communication and Signaling

Article Title: Structural maintenance of chromosome protein 1A exacerbates liver fibrosis by enhancing hepatic stellate cell activation and extracellular matrix synthesis via laminin subunit gamma 2 activation

doi: 10.1002/ccs3.70067

Figure Lengend Snippet: LAMC2 overexpression supports the profibrotic phenotype of HSCs in the presence of sh‐SMC1A. (A) The mRNA expression of LAMC2 in LX‐2 cells infected with sh‐SMC1A + NC‐oe or sh‐SMC1A + LAMC2‐oe was examined via RT‐qPCR ( n = 3). The expression of the profibrotic genes ACTA2 (B), TIMP1 (C), and PDGFRA (D) in LX‐2 cells was examined via RT‐qPCR ( n = 3). (E–G) The total protein content and phosphorylation of PI3K and Akt in LX‐2 cells were examined using Western blot assays ( n = 3). (H–J) Representative immunofluorescence images (H) and quantification of collagen I (I) and α‐SMA (J) in LX‐2 cells ( n = 3). (K‒M) The protein expression of LRAT and GFAP, which are markers of HSC activation, in LX‐2 cells was analyzed by Western blot assays ( n = 3). Graphical data are presented as means ± SDs. * p < 0.05, ** p < 0.01, and *** p < 0.001. An unpaired t ‐test or one‐way ANOVA was used for statistical analysis. ACTA2, actin alpha 2, smooth muscle; ANOVA, analysis of variance; HSCs, hepatic stellate cells; LAMC2, laminin subunit gamma 2; PDGFRA, platelet‐derived growth factor receptor alpha; RT‐qPCR, reverse transcription quantitative PCR; SMC1A, structural maintenance of chromosome protein 1A; TIMP1, TIMP metallopeptidase inhibitor 1.

Article Snippet: HSC LX‐2 cells (CL‐0560) were procured from Procell.

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Immunofluorescence, Activation Assay, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction