vector mscv transgene pgk puro ires gfp  (Addgene inc)

Journal: Cell Death & Disease

Article Title: The transcriptional repressor HDAC7 promotes apoptosis and c-Myc downregulation in particular types of leukemia and lymphoma

doi: 10.1038/cddis.2014.594

Figure Lengend Snippet: HDAC7 leads to the repression of c-Myc in SD-1 and Namalwa cells. ( a ) and ( b ) RT-qPCR validation for selected downregulated genes in the presence of HDAC7 are shown in SD-1-1-Tet-On-Tight HDAC7 ( a ) SD-1 and Namalwa-Tet-On-Tight HDAC7 cells ( b ). * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001. ( c ) TF-binding sites enriched in the downregulated genes after HDAC7 expression in SD-1 cells. Myc target genes are shown. ( d ) Western blot showing the downregulation of c-Myc after HDAC7 expression in SD-1 cells. ( e ) SD-1-Tet-On-Tight-HDAC7 and Namalwa-Tet-On-Tight-HDAC7 cells transduced with either MSCV-Empty or MSCV-c-Myc retroviral vectors were cultured and treated, or not, with doxycycline. At the indicated times after treatment, the cell number was assessed by cell counting. Trypan blue-dyed cells were omitted from the cell counts. Means±S.D. of the three independent experiments performed in triplicate. * P <0.05 ( f ) GSE34861 data were analyzed to determine any correlation between HDAC7 and c-MYC expression. c-MYC and HDAC7 probes were normalized with respect to values of healthy patients. The graph shows the negative correlation between c-MYC and HDAC7 (cds1 ρ= −2.15e-01; cds2 ρ= −2.09e-01) expression in the B-ALL patients

Article Snippet: MSCV-c-Myc-GFP retroviral vector was obtained from Addgene (Cambridge, MA, USA). pLKO.1-shHDAC3KD1-GFP and pLKO.1-shHDAC3KD2-GFP constructs were generated by cloning two validated shRNAs sequences that target HDAC3 (SIGMA, St. Louis, MO, USA), into the pLKO.1-GFP lentiviral vector.

Techniques: Quantitative RT-PCR, Binding Assay, Expressing, Western Blot, Transduction, Cell Culture, Cell Counting