Journal: The Journal of biological chemistry

Article Title: Activation of Nur77 by selected 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induces apoptosis through nuclear pathways.

doi: 10.1074/jbc.M500107200

Figure Lengend Snippet: FIG. 4. Nuclear localization of Nur77. A, immunostaining. Panc-28 cells were treated with Me2SO or 10 M Nur77 agonists for 6 h, and cells were immuno- stained for Nur77 as described under “Materials and Methods.” Nur77 staining was not observed in cells treated with nonspecific IgG. B, nuclear localization in subcellular fractions. Panc-28 cells were treated with the various compounds for 12 h and Nur77 protein expression in cy- tosolic, and nuclear extracts were deter- mined by Western blot analysis. Sp1 pro- tein and a nonspecific (NS) band serve as loading controls, and Sp1, a nuclear pro- tein, also serves as a control for separa- tion of nuclear and cytosolic extracts.

Article Snippet: Antibodies for PARP (sc8007), Sp1 (sc-59), and TRAIL (sc7877) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Nur77 (IMG-528) from Imgenex (San Diego, CA).

Techniques: Immunostaining, Staining, Expressing, Western Blot, Control

Journal: The FASEB Journal

Article Title: Global spliceosome activity regulates entry into cellular senescence

doi: 10.1096/fj.202000395rr

Figure Lengend Snippet: FIGURE 5 Sp1 is a key transcription factor of spliceosome regulation and cellular senescence. A, Genes positively or negatively correlated with the commonly regulated 58 spliceosomal genes were selected by correlation estimate > 0.7 or < −0.7, respectively, in RS and OSIS. Among the significantly correlated genes, 43 positively (left) and 25 negatively (right) associated TFs (Supplementary Table S4) were selected using human TF list (n = 1,267) obtained from DBD (http://www.transcriptionfactor.org). B, Transcription factors with binding potential for the 58 spliceosomal genes were identified using the DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). Bar plot indicates the −log10 (P-value) for 27 TFs which were listed in Supplementary Table S5. P-values indicate modified one-tail Fisher’s exact probability value used for gene-enrichment analysis. C, Association of the 27 TFs with the 58 spliceosomal genes was examined in RS and OSIS. Each point indicates the association of individual TFs. Sp1 has the strongest positive association with 17 target spliceosomal genes (Supplementary Table S5). D, Western blot using OSIS of HDF. E, Western blot using RS of HDF. F-I, HDFs (DT2) were transfected with siRNAs (50 nM) against Sp1 for 4 days. F, Western blot (left) and cell growth (right). Results are presented as mean + SD (n = 6). *P < .05; **P < .01 versus NC. G, SA-β-gal assay. Quantification of SA-β-gal (+) cells (upper) and representative cell images (lower panel) are shown. Results are presented as mean + SD (n = 6). **P < .01 versus NC. H, Western blots for SF proteins. I, mRNA levels for Sp1 and SFs using qRT-PCR. Results are presented as mean + SD (n = 6). **P < .01 versus NC. J, Potential Sp1 binding positions in the promoter regions (from −2000 to +1000) of HNRNPA3, HNRNPUL1, and SRSF7 were predicted as described in “Materials and Methods” and are marked as black ovals. The predicted PCR products and the designed primer sets were marked with blue arrow lines and # numbers (#1 to #3 or #4 for individual target gene). Green # numbers indicates the Sp1- binding regions confirmed in Figure 5L. Transcription start site for each gene was marked with red arrows and +1. K, L, Primary HDF (DT2) were used for ChIP assay as described in “Materials and Methods.” K, Western blot for ChIP bound Sp1. L, ChIP-qPCR. Fold enrichment was obtained by normalizing the arbitrary units obtained by qPCR for each target gene after ChIP using Sp1 antibody with the values using control IgG. Results are presented as mean + SD (n = 4). *P < .05 versus control IgG

Article Snippet: An aliquot was saved for input control and the other remaining aliquot was subjected to ChIP using Sp1 antibody (NB600-232, Novus Biologicals, Littleton, CO, USA) and Protein G Agarose/Salmon Sperm DNA (50% slurry, Millipore, Burlington, MA, USA).

Techniques: Binding Assay, Modification, Western Blot, Transfection, Quantitative RT-PCR, ChIP-qPCR, Control

Journal: Annals of oncology : official journal of the European Society for Medical Oncology

Article Title: Low expression of Mel-18 predicts poor prognosis in patients with breast cancer.

doi: 10.1093/annonc/mdq241

Figure Lengend Snippet: Figure 1. Immunohistochemical analyses of Mel-18 staining. As can be seen, Mel-18 shows strong nuclear staining in matched adjacent noncancer tissues (A, ·100; B, ·400), moderate cytoplasm staining in primary breast cancer tissues (C, ·100; D, ·400) and no positive staining for Mel-18 detectable in corresponding metastatic lymph node tissues (E, ·100; F, ·400) obtained from the same patients. Mel-18 shows increased expression in the adjacent noncancer tissue compared with that in the breast cancer tissues (H, ·100; G, I, ·400).

Article Snippet: Immunohistochemical kit (SP-9001 rabbit SP kit, lot: 50581654) was obtained from Zhongshan Golden Bridge Biotechnology Co. Ltd. (Beijing, China).

Techniques: Immunohistochemical staining, Staining, Expressing