Journal:

Article Title: Transcriptional Networks and Cellular Senescence in Human Mammary Fibroblasts D⃞

doi: 10.1091/mbc.E04-05-0392

Figure Lengend Snippet: Outline of the experimental conditions used for microarray comparisons. Primary breast fibroblasts (HMF3) were either immortalized with the catalytic subunit of human telomerase (hTERT) and wild-type LT antigen (LTwt), conditionally immortalized with hTERT and temperature-sensitive LT antigen (LTts), or passaged through to senescence. The HMF3Dwt cells have LTwt and continue to proliferate when cultured at 39°C. The HMF3A cells have LTts and become senescent after 7 d at 39°C. RNAi lines were created by introducing pSUPER vector constructs into HMF3A lines.

Article Snippet: Antibodies used were IL-1β, MKP-1, ID1, p21, p107, p130, GADD34, G-CSF, MEIS2, C/EBPα (C-18), C/EBPγ (E-19), and C/EBPβ (H-7) (Santa Cruz Biotechnology, Santa Cruz, CA); BUB1B (Exalpha Biologicals, Maynard, MA), HMGB2 (BD Biosciences PharMingen, San Diego, CA); and LT and p53 (PAb416 and 421( Harlow et al ., 1981 ).

Techniques: Microarray, Cell Culture, Plasmid Preparation, Construct

Journal:

Article Title: Transcriptional Networks and Cellular Senescence in Human Mammary Fibroblasts D⃞

doi: 10.1091/mbc.E04-05-0392

Figure Lengend Snippet: Differences in the RNAi lines. RT-PCR was used to determine the effectiveness of the stable RNAi vectors against BTG2, BTG3, NR4A3, and PHLDA1. (A) HMF3A cells containing the various p-SUPER vectors were shifted to 39°C for 7 d, and 3A-BTG2i cells also were shifted back to 33°C for 7 d. (B) The protein levels for MKP-1 (product of the DUSP1 gene) and IL-1β were examined for the various RNAi cultures after the cells were shifted to 39°C for 7 d. (C) The DNA binding activity of AP-1 was measured by EMSA after 7 d of LT inactivation in the DUSP1i and control cells. A cold competition (cc) control sample was used to assess the specificity of the band. (D) Staining of cells shifted to 39°C for 2 wk and then either shifted back to 33°C for 2 wk (back to 33°C) or left at 39°C for another 2 wk (39°C) was performed for 3A-STACβI, 3A-PHLDA1i, and control cells. (E) Comparison of the structure of STAC and the splice variant STACβ. RT-PCR were performed with various primer combinations (1–6). If STACβ has the same transcription start site as STAC, it would code for a shorter protein with the sequence given here, with the underlined area indicating where it would be different from STAC. For RNAi, a target sequence (+) was used that was only in STACβ. The resulting cells (39*) did have less STACβ mRNA than control cells, when compared after shifting of both cells to 39°C for 7 d. RT-PCR were performed three times with different RNA lysates. In addition to this samples were taken at three different times to ensure that the reaction was in the linear amplification stage. Westerns were performed at least twice by using different lysates. EMSA were performed three times by using different lysates.

Article Snippet: Antibodies used were IL-1β, MKP-1, ID1, p21, p107, p130, GADD34, G-CSF, MEIS2, C/EBPα (C-18), C/EBPγ (E-19), and C/EBPβ (H-7) (Santa Cruz Biotechnology, Santa Cruz, CA); BUB1B (Exalpha Biologicals, Maynard, MA), HMGB2 (BD Biosciences PharMingen, San Diego, CA); and LT and p53 (PAb416 and 421( Harlow et al ., 1981 ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay, Control, Staining, Comparison, Variant Assay, Sequencing, Amplification