Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1 promotes HSC recruitment in vitro and in vivo (A) Representative IHC immunostaining images showing staining of α-SMA-positive cells in SAA1-siRNA2, Neg-siRNA, and control samples obtained from CCl 4 and CI injury models. Bar graph represents quantification of IHC images per 5 fields. (B and C) Representative confocal immunofluorescence images showing co-localization of SAA1 and HSCs in SAA1-siRNA2, Neg-siRNA, and control in CCl 4 and CI injury models. (D and E) Transwell migration assay representing co-culture of primary mouse hepatocytes (PMHep) isolated from injured and healthy mouse with JS1 cells (D) and HepG2 cells (SAA1 overexpressing and GFP expressing) with LX-2 (E). The bar graph represents quantification of relative number of migrated cells. (F and G) Agarose spot (F) and Transwell (G) migration assays showing migration of LX-2 cells toward rhSAA1 at indicated time intervals. The bar graph represents quantification of relative number of migrated cells. (Scale bar = A, B, and C/200; G/100 μm: Inset/50 μm). Where applicable, data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and (n = 3). iPMHep represents injured primary mouse hepatocytes, hPMHep represents healthy primary mouse hepatocytes, HepG2 (SAA1) represents HepG2 cells overexpressing SAA1, and HepG2 (GFP) represents HepG2 expressing GFP.

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: In Vitro, In Vivo, Immunostaining, Staining, Immunofluorescence, Transwell Migration Assay, Co-Culture Assay, Isolation, Expressing, Migration

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: Screening of a receptor target for SAA1 in HSCs (A and B) LX-2 and PRHSCs were stimulated with rhSAA1 or rmSAA1, respectively, for 24 hr, and mRNA levels of TLR2, TLR4, FPR2, RAGE, and SR-B1 were determined by RT-qPCR. (C and D) Representative western blot analysis of receptors TLR2, TLR4, FPR2, RAGE, and SR-B1 after rhSAA1 and rmSAA1 treatment of LX-2 and PRHSCs, respectively. (E and F) LX-2 and PRHSCs were transfected with reporter plasmid for NF-κB. Twenty four hr later, the medium was changed and cells were pretreated with inhibitors of TLR2 (CU-CPT22, 1 μM), RAGE (FPS-ZM1, 0.5 μM), TLR4 (TAK 242, 5 nM), and FPR2 (WRW4, 0.25 μM) and then stimulated with rhSAA1 and rmSAA1, respectively, diluted in serum-free DMEM. Twelve hr later, luciferase activity was measured in fold induction after normalization with Renilla luciferase (Rluc) control. (G and H) Co-immunoprecipitation (co-IP) experiment showing the interaction between SAA1 and TLR2 determined by silver staining and western blot analysis. (For detailed information please see section). (n = 3). Where applicable, data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and (n = 3).

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Silver Staining

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: TLR2 serves as a chemotactic receptor for SAA1 and mediates migration of HSCs (A) Schematic of the TLR2 gene knockout strategy. The Cas9/sgRNA(s) target site(s) are indicated in red and were confirmed by sequencing. (B) Sequencing result of targeted region. (C) Confirmation of TLR2 gene KO at protein level determined by western blot. (D and E) WT and TLR2 −/− LX-2 cells (D) and WT and siPRHSCs (E) were transfected with reporter plasmid for NF-κB. Luciferase activity was measured as fold induction in comparison to untreated control (See also section for detail information). (F and G) Representative images showing agarose spot and Transwell migration assays of SAA1-treated WT LX-2, TLR2 −/− LX-2 cells, and non-treated control. (H and I) Representative images for immunostaining of TLR2 (H) and α-SMA + cells (I) in CU-CPT22- or VL-treated samples as shown in CCl 4 and CI injury models. Bar graph represents quantification of IHC images per 5 field(s). (J and K) Immunofluorescence images showing co-localization of SAA1 and HSCs in CU-CPT22-treated and control (VL) samples in CCl 4 (J) and CI injury models (K). (Scale bar represents 100 μm for CCl 4 injury, 200 μm for CI injury, and 50 μm for inset). Data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.0001 (n = 3). (For detailed TLR2 knockout strategy, see section).

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: Migration, Gene Knockout, Sequencing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Immunostaining, Immunofluorescence, Knock-Out

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis guides homing of transplanted LX-2 toward injury site(s) (A) Schematic representation of experimental design. (B) Bioluminescence imaging of transplanted cells entrapped to the liver 24 hr after transplantation in CCl 4 injury model, (A) represents sham operated mice, (B) represents SAA1siRNA2-treated mice transplanted with WT LX-2, (C) represents Neg-siRNA-treated mice transplanted with TLR2 −/− LX-2, and (D) represents control mice transplanted with WT LX-2 cells. (C and D) Representative confocal immunofluorescence images showing the recruitment of transplanted WT and TLR2 −/− LX-2 cells at injury locus after CCl 4 and CI injuries induced in SAA1-siRNA2, Neg-siRNA, and control samples, respectively. Bar graph represents quantification of relative number of homed cells at injury site(s). (Scale bar represents 100 μM). Data represent mean ± SEM ∗p < 0.01, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001(n = 3).

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: Imaging, Transplantation Assay, Immunofluorescence

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis induces Rac GTPase-mediated actin reorganization and migration of HSCs (A) Time course of GTPase-Rac1 activation(s) in LX-2 cells at indicated time interval (B and C) Basal and stimulated levels of GTPase-Rac1 in WT and TLR2 −/− cells as determined by pull-down assays (D and E) WT and TLR2 −/− LX-2 cells were pretreated with NSC 23766 (50 μΜ), and activation of GTP-Rac1 was determined by pull-down assay (D), and MLCK and p-MLC at Ser19 activity was determined by western blotting (E). (F and G) Migration assays showing pretreatment of the cells with NSC 23766 (50 μΜ) attenuated their migration(s) in agarose spot (F) and Transwell (G) assays. (H) Time course of PI3K activations after treatment of LX-2 cells at indicated time points. (I and J) WT and TLR2 −/− cells were pretreated with LY294002 (10 μΜ), and the phosphorylation of PI3K at p85 subunits was determined by western blot analysis, and the activations of Rac GTPase were determined by pull-down assay. (K and L) Migration assays showing pretreatment of the cells with LY294002 attenuated their migration(s) in agarose spot (K) and Transwell (L) assays. Photographs are representatives of (n = 6). (Scale bar represents 200 μm). Data represent mean ± SEM ∗p < 0.01 and ∗∗p < 0.001 (n = 3). Photographs are representatives of (n = 6). (Scale bar represents 200 μm). Data represent mean ± SEM ∗p < 0.01 and ∗∗p < 0.001 (n = 3).

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: Migration, Activation Assay, Pull Down Assay, Activity Assay, Western Blot

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis mediates increased deposition of ECM at injury sites and induces chemokines secretion in activated HSCs (A and B) Sirius red staining of ECM deposition at injury locus in CCl 4 and Cl-induced models. (C) ELISA detection of MCP-1, IL-8, and RANTES secretion from LX-2 cells after treatment of the cells with rhSAA1 for 24 hr. (D) mRNA levels of MCP-1, IL-8, and RANTES. (E) Protein-level detection of MCP-1, IL-8, and RANTES. (Scale bar represents 100 μm). Data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01 (n = 3).

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet:

Article Snippet: LX-2/human hepatic stellate cell line , Merck Millipore , Cat#SCC064.

Techniques: Marker, Plasmid Preparation, Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activation Assay, Silver Staining, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Transfection, Expressing, Reporter Assay, Staining, Over Expression, Software, Imaging, Live Cell Imaging, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease

doi: 10.1111/jcmm.13158

Figure Lengend Snippet: Pl GF is overexpressed in activated hepatic stellate cells ( HSC s) and its expression is induced by hypoxia dependent on HIF ‐1α. (A) Immunofluorescent expression of Pl GF (green) and α‐ SMA (red) in LX ‐2 human cell line and primary rat HSC s at different times in vitro activation; DAPI as blue nuclear counterstain. Scale bar = 100 μm for each picture. (B) The levels of Pl GF mRNA expression in rat HSC s at different times in vitro activation were examined by quantitative RT ‐ PCR ( n = 5). (C) The levels of Pl GF protein expression in rat HSC s at different time points in vitro activation were examined by Western blot. (D) Immunofluorescent expression of Pl GF (green) in rat HSC s; DAPI as blue nuclear counterstain. After infection with HIF ‐1α sh RNA (sh HIF ‐1α) or NTC sh RNA (sh NTC ), cells were cultivated under hypoxic conditions (1% O 2 ) or normoxia (21% O 2 ) for 24 hrs. Scale bar = 100 μm for each picture. (E) The levels of Pl GF mRNA expression in rat HSC s were measured by quantitative RT ‐ PCR . (F) Western blot analysis demonstrating effective silencing of HIF ‐1α on the expression of Pl GF in rat HSC , and GAPDH as loading control ( n = 3).

Article Snippet: LX‐2 human hepatic stellate cell line was from ScienCell Research Laboratory (Carlsbad, CA, USA) and was cultured as described previously .

Techniques: Expressing, In Vitro, Activation Assay, Quantitative RT-PCR, Western Blot, Infection, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease

doi: 10.1111/jcmm.13158

Figure Lengend Snippet: Pl GF knockdown by si RNA inhibits the proliferation and activation of hepatic stellate cells ( HSC s) via the PI 3K/Akt signalling pathway. (A) Measurement of cell proliferation of rat HSC s using CCK ‐8 assay. Cells were transfected with Pl GF si RNA or NTC si RNA ; cells were treated with rPlGF administration (50 ng/ml) or co‐incubation with PI 3K inhibitor LY 294002 for 5 days. a P < 0.001 compared with mimics control ( PBS ), b P < 0.001 compared with Pl GF + LY 294002. (B) The mRNA levels of α‐ SMA in rat HSC s with stimulation Pl GF (50 ng/ml) for 6 or 24 hrs. (C) Representative Western blot of α‐ SMA expression in rat HSC treated with rPlGF (50 ng/ml) or PBS for 6–24 hrs and quantification compared to β‐actin content. (D) Western blot for PI 3K, phospho‐Akt (p‐Akt), Akt and α‐ SMA in rat HSC s, and β‐tubulin or β‐actin as loading controls. Cells were treated with rPlGF administration (50 ng/ mL ) or co‐incubation with PI 3K inhibitor LY 294002 for 8 hrs. (E) The Western blot results of part (D) were quantified by densitometry.

Article Snippet: LX‐2 human hepatic stellate cell line was from ScienCell Research Laboratory (Carlsbad, CA, USA) and was cultured as described previously .

Techniques: Knockdown, Activation Assay, CCK-8 Assay, Transfection, Incubation, Control, Western Blot, Expressing