Journal: Blood cells, molecules & diseases
Article Title: A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression
Figure Lengend Snippet: The effects of knocking down HMI-LNCRNA on hemoglobin, MYB and HBS1L in HUDEP-2 cells ( A ) Illustration of timeline for transduction and culture of HUDEP-2 cells. Cells were maintained in expansion medium. ( B ) Expression level of HMI-LNCRNA was determined by qPCR in HUDEP-2 cells that were not transduced (naïve), and transduced with either scrambled shRNA or HMI-lncRNA shRNA lentiviruses by qPCR. ( C ) HBG, HBB, MYB and HBS1L transcript levels, and ( D ) percent HBG and HBB out of the total of both transcripts were measured in these same samples. ( E ) Protein expression of c-MYB, HBG and HBB were analyzed by Western blot in naïve HUDEP-2 cells, and cells transduced with scrambled shRNA and HMI-lncRNA shRNA (HUDEP-1 cells were used as control). GAPDH was used as loading control. ( F ) Naïve HUDEP-2 cells, and cells transduced with scrambled and HMI-lncRNA shRNAs were stained with anti-HBG antibody, followed by secondary antibody labeled with Alexa Fluor-594 (in red) and DAPI to stain nuclei (in blue), and imaged with a fluorescent microscope at 40X magnification. For all qPCR analyses , ACTB was used as the endogenous control. p-values: *
Article Snippet: Next, the shRNA template for HMI-LNCRNA was ligated to the lentivirus vector pGreenPuro (System Biosciences), upstream the EF1 promoter, and labeled HMI-lncRNA shRNA. pGreenPuro scrambled shRNA (System Biosciences) was used as the negative control.
Techniques: Transduction, Expressing, Real-time Polymerase Chain Reaction, shRNA, Western Blot, Staining, Labeling, Microscopy