Journal: PLoS ONE

Article Title: The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB

doi: 10.1371/journal.pone.0269735

Figure Lengend Snippet: Freshly prepared FBs were treated with (A) 10 ng/mL TGF-β or (B) 100 nM AngII, or (C) were subcultured. Cells lysates were then used for western blotting analysis. The expression levels of p53, phosphorylated p53 (p-p53 S15 or p-p53 S392), NFκB p65, phosphorylated NFκB p65 (p-NFκB p65), vimentin, and α-smooth muscle actin (α-SMA) were monitored by western blotting. GAPDH was used as a loading control. For each group, three independent cultures were prepared and treated (n = 3).

Article Snippet: The transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against α-SMA (Sigma-Aldrich, A5228), SMAD7 (Invitrogen), caspases (Antibody Sampler Kit, Cell Signaling), phospho–NFκB p65 (Ser 536) (Santa Cruz Biotechnology), NFκB p65 (Santa Cruz Biotechnology), cleaved PARP, PARP, phospho-IκBα, or IκBα (Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB

doi: 10.1371/journal.pone.0269735

Figure Lengend Snippet: Freshly prepared FBs were trans-differentiated to MyoFBs by treating them with 10 ng/mL TGF-β for 48 hours. MyoFBs were then cultured in control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for a further 48 hours in the presence or absence of 1 μM IKK VII, a selective IκB kinase inhibitor. (A) Cell lysates were used for western blotting analysis of NFκB p65, phosphorylated NFκB p65 (p-NFκB p65), IκB, phosphorylated IκB (p-IκB), caspase 3 (Cas3), cleaved caspase 3 (c-Cas3), caspase 7 (Cas7), cleaved caspase 7 (c-Cas7), Poly (ADP-ribose) polymerase (PARP), and cleaved PARP (cPARP). GAPDH was used as a loading control. (B) Cells were co-stained with TUNEL (green) and p53 (red). Hoechst dye was used for nuclear staining. TUNEL-positive apoptotic cells were counted and plotted. Scale bar is 50 μm. For each group, three independent cultures were prepared and treated (n = 3). *P<0.05, **P<0.01.

Article Snippet: The transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against α-SMA (Sigma-Aldrich, A5228), SMAD7 (Invitrogen), caspases (Antibody Sampler Kit, Cell Signaling), phospho–NFκB p65 (Ser 536) (Santa Cruz Biotechnology), NFκB p65 (Santa Cruz Biotechnology), cleaved PARP, PARP, phospho-IκBα, or IκBα (Cell Signaling Technology) overnight at 4°C.

Techniques: Cell Culture, Control, Western Blot, Staining, TUNEL Assay

Journal: PLoS ONE

Article Title: The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB

doi: 10.1371/journal.pone.0269735

Figure Lengend Snippet: Freshly prepared FBs were transfected with 25 nM SMAD7 siRNA and were trans-differentiated to MyoFBs by treating them with 10 ng/mL TGF-β for 48 hours. MyoFBs were then cultured in control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for a further 48 hours. (A) Cell lysates were used for western blotting analysis of SMAD7, p53, phosphorylated p53 (p-p53 S15 or p-p53 S392), NFκB p65, phosphorylated NFκB p65 (p-NFκB p65), IκB, phosphorylated IκB (p-IκB), caspase 3 (Cas3), cleaved caspase 3 (c-Cas3), caspase 7 (Cas7), cleaved caspase 7 (c-Cas7), Poly (ADP-ribose) polymerase (PARP), and cleaved PARP (cPARP). GAPDH was used as a loading control. (B) Cells were co-stained with TUNEL (green) and p53 (red). Hoechst dye was used for nuclear staining. TUNEL-positive apoptotic cells were counted and plotted. Scale bar is 50 μm. For each group, three independent cultures were prepared and treated (n = 3). *P<0.05, **P<0.01.

Article Snippet: The transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against α-SMA (Sigma-Aldrich, A5228), SMAD7 (Invitrogen), caspases (Antibody Sampler Kit, Cell Signaling), phospho–NFκB p65 (Ser 536) (Santa Cruz Biotechnology), NFκB p65 (Santa Cruz Biotechnology), cleaved PARP, PARP, phospho-IκBα, or IκBα (Cell Signaling Technology) overnight at 4°C.

Techniques: Transfection, Cell Culture, Control, Western Blot, Staining, TUNEL Assay

Journal: International journal of oncology

Article Title: Quinone methide tripterine, celastrol, induces apoptosis in human myeloma cells via NF-κB pathway.

doi: 10.3892/ijo.2011.1161

Figure Lengend Snippet: Figure 5. Effects of celastrol on NF-κB activity in myeloma cells. (A) U266 cells were treated with 50 ng/ml TNF-α for 12 h, and then the cells were incubated with 0.5 µM of celastrol for 6 and 12 h. The DNA binding activity of NF-κB in U266 cells was quantified by ELISA with the use of a Trans-AM NF-κB p65 transcription factor assay kit. Celastrol significantly suppressed the stimulatory effect of TNF-α on NF-κB DNA binding activity (P<0.01). The control is referred to as no stimulated control, the wild-type as wild-type oligonucleotide, and the mutant as mutant oligonucleotide. (B) Effects of celastrol on the constitutive expression of NF-κB and IκB-α in myeloma cells. U266 cells were treated with celastrol (0.5 µM) for the indicated times. Cell lysates (15 mg protein per lane) were fractionated in 12.5% SDS-polyacrylamide gels and analyzed by Western blotting with an antibody against NF-κB p65 and IκB-α. Nuclear and cytoplasmic extracts were prepared in order to check these proteins by Western blotting.

Article Snippet: The level of NF-κB was assessed by ELISA using monoclonal antibodies and the procedure recommended by the manufacturer (TransAM NF-κB p65 transcription factor assay kits; Active Motif, Carlsbad, CA).

Techniques: Activity Assay, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Transcription Factor Assay, Control, Mutagenesis, Expressing, Western Blot