XB-IMG-124937
Xenbase Image ID: 124937
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Figure 5. Effects resulting from inhibition of GEMC1 expression in mammalian cellsa) RT-PCR of mouse GEMC1 (mGEMC1) and GAPDH (GAPDH) mRNA derived from mouse NIH 3T3 fibroblasts that were treated with control (Mock) or mouse GEMC1 siRNA oligos (mGEMC1_1 and mGEMC1_2). b) Quantification of RNA levels shown in (a). The graph shows data from one representative experiment. c) Histograms relative to FACS analysis following incorporation of BrdU in mouse NIH3T3 cells that were untreated (Un), mock treated (Mock), transfected with two independent mouse GEMC1 siRNA (mGEMC1_1 or mGEMC1_2) or with Cdc45 siRNA oligos. The graph shows data from one representative experiment. d) Typical FACS profiles relative to experiments shown in (c). Circles in pink indicate BrdU incorporating cells. e) WB of MEF-3T3 cells stably transfected with empty TET-OFF vector (Vector), with TET-OFF vector expressing hemagglutinin epitope (HA) tagged mGEMC1 (mGEMC1) or with the same vector expressing siRNA resistant HA tagged mGEMC1 (mGEMC1* Rescue). Cells were subjected to silencing with mGEMC1_1 siRNA in the absence or presence of 1 μg/ml doxycycline (Dox), which represses the expression driven by the exogenous promoter present on the vector. After 96 hours cells were collected and processed for WB using anti HA and anti GAPDH antibodies as indicated. f) The histograms represent the percentage in S-phase of MEF-3T3 cells that were treated with mock (Mock) or mGEMC1 siRNA oligos (mGEMC1_1 siRNA). mGEMC1_1 siRNA treated cells were stably transfected with empty TET-OFF vector (Vector), with TET-OFF vector expressing HA tagged mGEMC1 (mGEMC1) or with the same vector expressing siRNA resistant HA tagged mGEMC1 (mGEMC1*) in the absence (−Dox) or in the presence (+Dox) of 1 μg/ml doxycycline. Cells were BrdUTP labelled and processed for FACS analysis. The graph shows data from one representative experiment. g) Model for GEMC1 function at replication origins (see text for explanation). See Supplementary Fig 6 for uncropped gels shown in this figure. Image published in: Balestrini A et al. (2010) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View |