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Percentage of cells in each cell cycle phase by flow cytometry for knockdown of NET4/TMEM53 and controls in U2OS cells. The siRNAs were transfected 24 h after transfection of NET4/Tmem53-GFP in order to enable protein to be generated from the plasmid prior to the beginning of knockdown. Because antibodies to NET4/Tmem53 recognized multiple bands on Western blot at the expected molecular weight, knockdown of the protein was tested using a NET4/Tmem53-GFP fusion and GFP antibodies. (D) Western blot demonstrating the knockdown of NET4/Tmem53. PPIA was used as a control for normalization. (C) NET4/TMEM53 transcript levels were also knocked down in the U2OS cell line using the siRNA oligo TMEM53 si2. Peptidylprolyl isomerase A (PPIA) was used as a control for normalization. (B) NET4/TMEM53 transcript levels were effectively knocked down in MRC5 cells using both siRNA oligos and an esiRNA. (A) Splice variants and position of knockdown oligos. Knockdown of NET4/TMEM53 in U2OS and MRC5 cells.
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(C) Because siRNAs failed to knock down NET4/TMEM53 in HEK293T cells, the cell cycle/DNA content profile effect of the original NET4/TMEM53-mRFP construct was tested on U2OS and MRC5 cells as alternatives for knockdowns. (B) Both long (l) and short (s) splice variants had the same effect on cell cycle/DNA content profiles and effects were independent of whether the GFP tag was on the amino terminus or carboxyl terminus. (A) Two splice variants of NET4/TMEM53 were cloned that differed only by a 33 amino acid insertion roughly a third of the way into the protein. Splice variants of NET4/TMEM53 and the effect of knockdown in U2OS and MRC5 cells. The pixel intensity for untransfected controls internal to each micrograph was set to 1 and average relative values from 40 transfected cells and 40 untransfected cells for each transfection are shown with standard errors. The graph on the right shows quantification of the pixel intensity for the phospho-pRb staining. All micrographs were taken at the same exposure time. In each panel two adjacent cells are marked by arrowheads, one transfected and the other not transfected. NET/mRFP signal is shown in the left panels to identify transfected cells and phospho-pRb staining in the right panels. Cells were stained with an antibody that recognizes pRb phosphorylated on serine 780.
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(B) Photomicrographs of cells overexpressing the RFP alone control or NET4/Tmem53. The knockdown of pRb is confirmed in the lower left corner. The 2N accumulation effect of NET4/Tmem53 on the cell cycle profile observed in HEK293T cells was lost when pRb levels were reduced. Cell cycle profiles are shown for these cells expressing NET4/Tmem53 or controls of mRFP alone or NET51/C14orf1 that had no effect in the original flow cytometry screen. (A) pRb was knocked down by siRNA in HEK293T cells subsequently transfected with NETs. PRb is also involved in NET4/Tmem53 effects. The results for the HEK293T cells are shown above those for the HCT116 p53−/− cells. (B) The percentage of cells in the 4N and 2N populations were calculated and 4N∶2N ratios were plotted from at least three separate experiments with standard errors shown. Most NETs that had produced increases in the 4N population in Figure 1 yielded similar increases in the 4N population in HCT116 p53−/− cells however, NET4/Tmem53 and NET59/Ncln lost their effects. (A) Flow cytometry profiles of cells expressing NET-mRFP fusions in HCT116 p53−/− cells. Images of HEK293T cells expressing NET-mRFP shows nuclear morphology depicted by DAPI staining (upper panel) compared to mRFP signal (lower panel) indicating cells expressing NETs.Ĭell cycle effects of NET4/Tmem53 and NET59/Ncln depend on p53. The changes in flow cytometry profile of cells expressing NETs are not caused by aberrant nuclear morphology of cells. Percentage of cells in each cell cycle phase by flow cytometry upon exogenous expression of NET-mRFP fusions. Arrows indicate significant changes in cell cycle profile between transfected and non-transfected cells. The transfected and untransfected populations were both set on the scale to 100 for the 2N population so that increases or decreases in the 4N peak reveal changes in the cell distribution. The red line is the mRFP expressing cells in the population while the blue line is the untransfected cells in the population (the majority of cells were not transfected).
FLOWJO 10 NORMALIZATION HISTOGRAM SOFTWARE
Data were analyzed using FlowJo software and histogram overlays are displayed as %Max, scaling each curve to mode = 100%. HEK293T cells expressing mRFP-NET fusions were recovered by trypsinization and analyzed by flow cytometry at 48 h post-transfection. Changes in flow cytometry cell cycle profiles for cells overexpressing NETs.