Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Hepatocytes were isolated and plated as described in Materials and Methods and treated with 25 to 200 μM DCA or no addition (control) for 24 h. ( A ) Real-Time RT-PCR analysis of miR-21 (n = 7). ( B ) Immunoblotting of PDCD4 (top; n = 7) and ratio between Wt and Mut miR-21 luciferase activity (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. Cells were co-transfected with a reporter vector consisting of a luciferase cDNA fused to the 3′ UTR of PDCD4, containing either a Wt or Mut miR-21 binding site. The cytomegalovirus- Renilla luciferase vector was used as an internal standard control. ( C ) Cell viability, measured by the ApoTox-Glo TM Triplex assay (top; n = 5), cell death measured by the LDH assay (middle; n = 7) and caspase-3/7 activity (bottom; n = 5). ( D ) Apoptotic cells were detected by Hoechst staining. Representative images of control and 25, 50, 100 and and 200 μM DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Isolation, Control, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Lactate Dehydrogenase Assay, Staining

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Primary rat hepatocytes were transfected with a miR-21 precursor (Pre-miR-21) or control (Pre-miR-C) and treated with 100 μM DCA or no addition for 24 h. ( A ) Real-Time RT-PCR analysis of miR-21 expression (n = 7). ( B ) Immunoblotting of PDCD4 (n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. ( C ) Cell viability (top), cytotoxicity (middle) and caspase-3/7 activity (bottom) measured by the ApoToxGlo TM Triplex assay (n = 5). Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Primary rat hepatocytes were transfected with a specific siRNA against PDCD4 (siRNA PDCD4) or a control (siRNA C) and treated with 100 μM DCA or no addition for 48 h. ( A ) Immunoblotting of PDCD4 (n = 4). Representative blots are shown. Blots were normalized to endogenous β-actin. ( B ) Cell viability, measured by the ApoTox-Glo TM Triplex assay (n = 4). ( C ) General cell death measured by LDH assay (n = 11). ( D ) Apoptotic cells detected by Hoechst staining. Representative images of control, DCA, PDCD4 silencing and PDCD4 silencing + DCA are shown. Bar, 30 μM. Arrows indicate apoptotic nuclei. Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Transfection, Control, Western Blot, Lactate Dehydrogenase Assay, Staining

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Cells were transfected with a plasmid encoding a constitutively active form of IKK (CA-IKK), NF-κB or an empty vector (pcDNA 3.1) or incubated with 10 μM BAY 11-7085 and treated with 100 μM DCA or no addition as described in Materials and Methods. ( A ) NF-κB transcriptional activity (n = 6). Cells were transfected with a mixture of an inducible NF-κB responsive construct, encoding the firefly luciferase reporter gene, and a constitutively expressing Renilla luciferase construct, as an internal standard control. ( B ) Real-Time RT-PCR analysis of miR-21 (top; n = 5) and immunoblotting of PDCD4 (bottom; n = 5). Representative blots are shown. Blots were normalized to endogenous β-actin. ( C ) Cell viability, measured by the ApoTox-Glo TM Triplex assay (n = 5). ( D ) NF-κB transcriptional activity (n = 4). ( E ) Real-time RT-PCR analysis of miR-21 (top; n = 6) and immunoblotting of PDCD4 (bottom; n = 8). Representative blots are shown. Blots were normalized to endogenous β-actin. ( F ) Cell death, measured by LDH assay (top; n = 8) and caspase-3/7 activity measured by the ApoTox-Glo TM Triplex assay (bottom; n = 6). Results are expressed as mean ± SEM fold change.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Transfection, Plasmid Preparation, Incubation, Activity Assay, Construct, Luciferase, Expressing, Control, Quantitative RT-PCR, Western Blot, Lactate Dehydrogenase Assay

Journal: Scientific Reports

Article Title: Inhibition of NF-κB by deoxycholic acid induces miR-21/PDCD4-dependent hepatocellular apoptosis

doi: 10.1038/srep17528

Figure Lengend Snippet: Wistar male rats (n = 6) were treated with 125 mg/kg/day of DCA by oral gavage for 1 and 3 days before sacrifice. Rats in the control group (n = 6) were administered with water as described in Material and Methods. ( A ) Real-Time RT-PCR analysis of miR-21. Results are expressed as mean ± SEM fold change. ( B ) Representative blots and quantitation, in fold-change from controls, of PDCD4, IκB/NF-κB ratio, PIDD-CC and active caspase-2 from liver of rats treated with DCA for 1 day (left) and 3 days (right). Blots were normalized to endogenous β-actin.

Article Snippet: Upon transference onto a nitrocellulose membranes and blocking with 5% milk solution, membranes were probed overnight at 4 °C with primary rabbit polyclonal antibodies against IκB (sc-371), NF-κB p65 (sc-372) and PIDD (sc-67032) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), primary rabbit polyclonal antibody against active caspase-2 (ab2251; Abcam PLC, Cambridge, United Kingdom), primary goat polyclonal antibody against PDCD4 (sc-27123; Santa Cruz Biotechnology) or primary mouse monoclonal antibodies against β-actin (A5441; Sigma-Aldrich Co.), HDAC1 (05–100; Upstate Biotechonology, Lake Placid, NY, USA), GAPDH (sc-32233; Santa Cruz Biotechnology) and, finally, with secondary antibodies conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at room temperature.

Techniques: Control, Quantitative RT-PCR, Quantitation Assay