Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 1 Plin5 is highly expressed in quiescent HSC and dramatically reduced in HSC on activation in vitro and in vivo. (a,b) Samples from an obese and fibrotic mouse model fed a regular chow diet (Chow) or a HFD. (a). Liver sections were stained with hematoxylin and eosin (left panels), Sirius Red (Central panels), or immuno-stain of α-SMA (right panels). Representative views were presented (b). Real-time PCR assays (n = 3). *Po0.05 vs HSC from the chow group. (c,d) Freshly isolated HSC from normal mice were cultured for 1 day (Day 1) or 7 days (Day 7). (c) Real-time PCR assays (n = 3). *Po0.05 vs HSC at Day 1. (d) Immuno-fluorescent stain of Plin5. Nuclei were stained by DAPI. The negative control (Neg ctr) was conducted without the primary anti-Plin5 antibodies. The inserted squares denoted the enlarged cells in the views. Representative views were presented.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Activation Assay, In Vitro, In Vivo, Staining, Immunostaining, Real-time Polymerase Chain Reaction, Isolation, Cell Culture, Negative Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 2 The expression of exogenous Plin5 inhibits HSC activation. Culture-activated HSC (4–9 passages in culture) were transfected with the recombinant lentiviral LV-Plin5-YFP, or the vehicle lentiviral LV-YFP, or with no transduction (Mock HSC). *Po0.05 vs mock HSC. Representatives were presented from three independent western blotting analyses. β-tubulin was used as an invariant control for equal loading. (a) Real-time PCR assays (n = 3); (b) western blotting analyses; (c) cell proliferation assays (ie, MTS assays) (n = 3); (d) real-time PCR assays (n = 3); (e) western blotting analyses.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Expressing, Activation Assay, Transfection, Recombinant, Transduction, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 3 Exogenous Plin5 restores the formation of LD and elevates cellular lipid content in activated HSC. Passaged HSC were transducted with LV- Plin5-YFP, or LV-YFP, or no transduction (Mock HSC) (a,c,d), or transducted with recombinant lentiviruses with different fragments of Plin5 cDNA, which expressed truncated domains of Plin5 (b). After selection with puromycin, cells were cultured in media with PA at 20 μM for 24 h. (a,b) After fixation, LDs in HSC were stained with Oil Red O. Representative views were presented. (c) Analyses of cellular FFA (n = 3), *Po0.05 vs mock HSC. (d) Analyses of cellular TG (n = 3). *Po0.05 vs mock HSC.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Transduction, Recombinant, Selection, Cell Culture, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 4 The expression of exogenous Plin5 regulates major modulators for lipid homeostasis and expression of genes related with lipid metabolism in HSC. (a) Passaged HSC were transducted with LV-Plin5-YFP (LV-Plin5) or LV-YFP. After selection with puromycin, these cells were respectively transfected with the plasmid PPRE-Luc, LXR-Luc, or Topflash. Luciferase activity assays were conducted (n = 6). *Po0.05 vs HSC transducted with LV-YFP. (b–d) Passaged HSC were transducted with LV-Plin5-YFP (LV-Plin5), or LV-YFP, or no transduction (Mock HSC). After selection of positively transducted cells with puromycin, total RNA was prepared for real-time PCR assays (n = 3). *Po0.05 vs mock HSC.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Expressing, Selection, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Transduction, Real-time Polymerase Chain Reaction

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 5 The expression of exogenous Plin5 dramatically reduces the level of ATGL and abhd5 in HSC. Passaged cells were transducted with LV-Plin5- YFP (LV-Plin5), or LV-YFP, or no transduction. After selection, total RNA and whole-cell extracts from the cells were prepared for real-time PCR assays (a) and western blotting analyses (b). *Po0.05 vs mock HSC (n = 3). Representatives were presented from three independent western blotting analyses. β-actin was used as an invariant control for equal loading.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Expressing, Transduction, Selection, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 8 The expression of exogenous Plin5 attenuates cellular oxidative stress in HSC. Passaged HSC were transducted with LV-Plin5-YFP (LV-Plin5), or LV-YFP, or no transduction (Mock). After selection with puromycin, cellular oxidative stress in these cells were analyzed (n = 3). *Po0.05 vs mock cells. (a) Cellular GSH assays; (b) analyses of the ratio of GSH to GSSG; (c) cellular LPO assays; (d) cellular ROS assays.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Expressing, Transduction, Selection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 7 The activity of AMPK regulates cellular lipid content in HSC. Passaged HSC were transducted with LV-Plin5-YFP or LV-YFP. After selection with puromycin, HSC were treated with the AMPK inhibitor Compd C (a,b), or the AMPK activator AICAR (c,d), at indicated concentrations for 24 h. *Po0.05 vs cells with LV-Plin5-YFP, but with no treatment (n = 3) (the corresponding left second column). (a,c) Cellular FFA assays; (b,d) cellular TG assays.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Activity Assay, Selection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 9 The expression of exogenous Plin5 induces gene expression of GCL subunits in HSC, likely by stimulating the transcriptional activity of Nrf2. Passaged HSC were transducted with LV-Plin5-YFP (LV-Plin5), or LV-YFP, or no transduction (Mock). After selection with puromycin, some of the cells were transfected with the plasmid p8xARE-Luc for luciferase assays. Cells were cultured for additional 24 h. (a). Real-time PCR assays. *Po0.05 vs mock cells (n = 3); (b) western blotting analyses. Representatives were presented from three independent experiments. β-tubulin was used as an invariant control for equal loading; (c) luciferase activity assays. *Po0.05 vs mock cells (n = 6). The floating schema denoted the plasmid p8x-ARE-Luc in use for transfection to the system.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Expressing, Gene Expression, Activity Assay, Transduction, Selection, Transfection, Plasmid Preparation, Luciferase, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Perilipin 5 restores the formation of lipid droplets in activated hepatic stellate cells and inhibits their activation.

doi: 10.1038/labinvest.2016.53

Figure Lengend Snippet: Figure 10 A simplified action model for Plin5 to inhibit the activation of HSC. Expression of Plin5 is depleted in HSC on activation. Introduction of exogenous Plin5 in HSC by transduction of LV-Plin5-YFP (LV-Plin5) restores the formation of cellular LDs in HSC and stimulates the activation of AMPK, which, in turn, induces the expression of endogenous Plin5. The activation of AMP results in the elevation of cellular lipid content in HSC by inducing lipogenesis and suppressing lipolysis. On the other hand, the activation of AMPK stimulates the transcription activity of Nrf2 and the expression of GCL subunits, leading to the elevation of cellular levels of GSH and the attenuation of cellular oxidative stress in HSC. These effects collectively inhibit HSC activation.

Article Snippet: The experiments were conducted as we previously described,21 using goat polyclonal primary antibody against Plin5 (1:50, sc-240627, Santa Cruz, CA, USA) and donkey anti-goat IgG secondary antibody conjugated with fluorescent Texas Red dye (1:300, cat#705-075-147, Jackson ImmunoResearch, PA, USA).

Techniques: Activation Assay, Expressing, Transduction, Activity Assay