dependent redistribution of nucleolar p14ARF.Figure 2A, B: HeLa cells were transfected with empty vector (e.v., control) or Myc-tagged wild type PRMT1-containing plasmid. Immunofluorescence (IF) staining was performed using α-p14ARF (red, endogenous p14ARF), α-Myc (green, exogenous PRMT1) antibodies, DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin). In (A), representative IF results are shown, with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo-/cytoplasmic p14ARF localization. The Merge displays the combination of p14ARF and PRMT1 staining. Scale bars: 15 μm. The quantification of exclusive nucleolar p14ARF was performed by cell counting (percentage of cells) and is shown for three independent experiments in (B) (mean ± SD). C, D: U2OS cells were transfected with EGFP-tagged p14ARF alone or in combination with Myc-tagged wild type PRMT1-containing plasmids. p14ARF-EGFP (green), DAPI (blue, nuclei/DNA) and PRMT1 (red, using α-Myc antibody) were visualized by fluorescence microscopy, for which representative results are shown in (C), with the lower images displaying a magnification as indicated in the upper images by the rectangles. Scale bars: 10 μm. The subcellular distribution of p14ARF-EGFP (exclusively nucleolar or not-exclusively nucleolar but predominantly nucleo-/cytoplasmic) was quantified in p14ARF-positive and p14ARF/Myc-PRMT1-double positive cells by cell counting (percentage of cells) for three independent experiments in (D) (mean ± SD, ***: p-value ≤ 0.001 using Welch´s t-test). E: U2OS cells were transfected with EGFP-tagged p14ARF alone or in combination with Myc-tagged wild type (wt) or catalytically inactive (mut) PRMT1-containing plasmids. The cellular distribution of p14ARF-EGFP was quantified as in (D). The relocalization of p14ARF out of the nucleolus upon overexpression of PRMT1 (wt or mut) was determined by cell counting (percentage of cells) for four independent experiments (mean ± SD, *: p-value ≤ 0.05 using the paired t-test). HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and three PRMT1-specific siRNAs). PRMT1 depletion was verified by immunoblotting using α-PRMT1 and α-β-TUBULIN (loading control) antibodies ((F); depicted staining results derive from the same blot as well as exposure times with white lines indicating where tracks were cut). HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and three PRMT1-specific siRNAs). The cellular p14ARF distribution was determined by immunofluorescence staining (IF) using α-p14ARF antibody. Representative IF images for the siControl_1 and siPRMT1_1 condition are shown in (G) (endogenous p14ARF in red and the merge additionally with DAPI in blue for nuclei/DNA). Scale bars: 15 μm. HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and three PRMT1-specific siRNAs). The subcellular localization of p14ARF (exclusively nucleolar or not-exclusively nucleolar but predominantly nucleo-/cytoplasmic) was quantified by cell counting (percentage of cells) for all used siRNAs in (H) and for siControl_1 / siPRMT1_1 from three independent experiments in (I) (mean ± SD, ***: p-value ≤ 0.001 using Welch´s t-test).