Journal: Oncotarget

Article Title: MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells

doi: 10.18632/oncotarget.11337

Figure Lengend Snippet: ( A ) Changes in morphology and gene expression in MZF-1 60– 72 construct-transfected stably cloned MB-231 cells. β-actin was used as a control, and c-Myc was used as a marker for transfected cells. ( B ) Confocal microscopy showing the distribution of the Elk-1 and MZF-1 proteins. Cells were stained with antibodies against Elk-1 and MZF-1, followed by the appropriate FITC- or rhodamine-conjugated secondary antibodies. Confocal slices of 0.5 and 0.6 μm thicknesses were obtained, and images were obtained focusing the center of the nucleus. “N” denotes the nucleus; “C” denotes the cytosol. ( C ) ChIP assays indicating the binding activity of endogenous Elk-1 and MZF-1 to the PKCα promoter in various cells. The assays were performed using an MZF-1 (left panel) or Elk-1 (right panel) antibody. ( D ) Visualization and quantification of cell migration and proliferation of modified MB-231 cells. ( E ) Tumor growth in nude mice after xenografts of modified MB-231 and Hs578T cells (left panels). Tumors removed from the mice were weighed (middle images and graph) and sliced before histological examination (right images). ** p < 0.01 compared with the empty vector-transfected MB-231 group, ## p < 0.01, compared with the empty vector-transfected Hs578T group.

Article Snippet: The cells were then fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.1% saponin, 2% goat serum (Vector Laboratories) and 0.02% NaN3 for 15 min. Elk-1 and MZF-1 were visualized using their particular antibodies (1:200) followed by FITC-conjugated goat anti-mouse IgG antibody and rhodamine-conjugated goat anti-rabbit IgG antibody (Rockland Immunochemicals, Inc., Gilbertsville, PA.), respectively.

Techniques: Gene Expression, Construct, Transfection, Stable Transfection, Clone Assay, Control, Marker, Confocal Microscopy, Staining, Binding Assay, Activity Assay, Migration, Modification, Plasmid Preparation

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 1. TNF or LPS activates IKK. (A) DPSCs were treated with TNF (10 ng/mL) and P. gingivalis LPS (1 g/mL) for the indicated times. Fifty-g aliquots of protein extracts were probed with polyclonal antibodies against IB (1:1000) or monoclonal antibodies against phospho-specific antibodies (1:1000). For loading control, the blots were stripped and re-examined with monoclonal antibodies against - tubulin (1:15, 000). (B) E. coli LPS activates IKK. DPSCs were treated with E. coli LPS (200 ng/mL) or TNF for the indicated time points. Western blot analysis was performed as described in (A).

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Bioprocessing, Control, Western Blot

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 2. TNF or LPS induces the nuclear translocation of NF-B. (A) DPSCs were treated with TNF (10 ng/mL) for the indicated time. Five- g aliquots of nuclear proteins were incubated with 32P-labeled NF-B oligonucleotide probes. For the supershift assay, nuclear proteins were pre-incubated with polyclonal antibodies against p65 for 10 min and then probed with32P-labeled NF-B oligonucleotide probes. (B) DPSCs were treated with P. gingivalis LPS (1 g/mL) for the indicated times. EMSA was performed as described in (A).

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Translocation Assay, Incubation, Labeling

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 3. LPS induces IL-8 expression in DPSCs. (A) DPSCs were treated with TNF or P. gingivalis LPS for the indicated times. Fifty-g aliquots of protein extracts were probed with polyclonal antibodies against phospho-specific p65. For loading control, the membrane was stripped and re-probed with polyclonal antibodies against p65. (B) DPSCs were treated with TNF for the indicated times. Ten-g aliquots of total RNAs were hybridized with 32P-labeled IL-8 cDNA probes. For loading control, the blot was stripped and re-probed with 32P-labeled glyceraldehyde-3- phosphate dehydrogenase (GAPDH) cDNA probes. (C) DPSCs were treated with P. gingivalis LPS for the indicated times. Northern blot analysis was performed as described in (B). (D) DPSCs were treated with TNF for the indicated times. Total RNAs were probed with 32P-labeled IL- 6 cDNA probes. (E) DPSCs were treated with E. coli LPS for the indicated times. Total RNAs were probed with 32P-labeded IL-6 cDNA probes.

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Expressing, Control, Membrane, Labeling, Northern Blot