Sequence conservation and expression of hRif1. (A) Schematic representation of sequence similarities of Rif1 proteins. Percentage identities are in black and percentage similarities are in red. A putative NLS is marked in green. The alternatively spliced-out peptide of hRif1 is marked in yellow. (B) Ubiquitous expression of hRif1 mRNA in human cancer cell lines. 2 μg of mRNAs from different cell lines were treated with glyoxal, electrophoresed on a 20 cm × 25 cm 0.8% agarose gel, transferred to a Hybond NX membrane and hybridized with hRif1 or β-actin cDNA probes. The bracket and arrowhead indicate groups of alternatively spliced hRif1 mRNAs. (C) Expression of hRif1 mRNA in adult human tissues. A multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was hybridized with hRif1 or β-actin cDNA probes. Separation of alternatively spliced hRif1 mRNA was not as clear as in B because the mRNA electrophoresis was performed in a much smaller (6 cm × 8 cm) 1% agarose gel. The bracket and arrowhead indicate the groups of hRif1 mRNAs corresponding to those indicated in B. (D) Immunoblot of hRif1 protein with antibody PAB2857. LOX cells were infected with lentiviruses expressing siRNA sequences targeting hRif1 mRNA. 3 d after infection, 50 μg of whole cell extracts were analyzed by Western blot analysis and probed with PAB2857.

No detectable localization of hRif1 protein at telomeres in normal cells or in cells depleted of hRap1, TRF1, or TRF2 protein. (A) Western blot analysis of whole cell extracts from LOX cells treated with lentiviruses expressing hairpin siRNAs against TRF1, TRF2, or hRap1. (B) Immunofluorescence analysis of hRif1 protein in LOX melanoma cells. LOX cells were infected with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, or TRF2. 3 d after infection, cells were fixed and stained with hRif1 antibody PAB2857 (red) and TRF1 or TRF2 antibodies (green). A microscope (model Eclipse E600; Nikon) with a 100× objective and a Coolsnap fx charge-coupled device camera and software (Roper Scientific) was used to visualize the image. (C) Histogram showing the average numbers of hRif1 foci per nucleus in LOX cells after treatment with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, TRF2, or with lentiviruses expressing hTER template mutant 47A. For each treatment, hRif1 foci in ∼50 interphase cells were counted after immunofluorescence analysis and the average numbers per nucleus were presented.

hRif1 localizes on uncapped telomeres without a requirement for wild-type p53. HCT116 cell lines with the p53 locus wild type or disrupted, but which were otherwise isogenic, were infected with lentiviruses expressing 47A-hTer. 5 d after infection, cells were fixed and stained with 53BP1 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described for Fig. 4. Bars, 10 μm.

Accumulation of hRif1 protein at DNA damage foci induced by MMS treatment. Images were acquired with a Deltavision microscopy system as described for Fig. 4. (A) LOX cells were treated with 0.01% MMS for 1 h, then fixed and immunostained with 53BP1 antibody (green) to visualize DNA damage foci, hRif1 antibody PAB2852 (red) to visualize the endogenous hRif1 protein, and DAPI (blue). (B) GFP-hRif1 fusion protein localizes to DNA damage foci induced by MMS treatment. LOX cells were transfected with pEGFP-hRif1 expression construct. 2 d after transfection, cells were treated with 0.01% MMS for 1 h, fixed and stained with 53BP1 antibody (red) and DAPI (blue). Bars, 10 μm.

hRif1 protein aligns along a subset of midzone microtubules at anaphase. (A) Immunostaining of hRif1 in early anaphase and late anaphase LOX cells using hRif1 antibody (red), β-tubulin antibody (green), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described in Fig. 8. (B) Enlarged images of early anaphase cells in A to show the alignment of hRif1 along microtubules. (C) Schematic representation of hRif1 deletion constructs used for analyses of telophase chromosome association and anaphase midzone microtubule colocalization. GFP-hRif1 deletion constructs were transfected into LOX cells and immunostaining was performed to determine their intracellular localization across the cell cycle. Bars, 10 μm.

Impaired cell growth upon depletion of hRif1. (A) Knockdown of hRif1 mRNA levels by siRNA treatment. LOX cells were infected with lentiviruses expressing hairpin siRNAs against different sequences within the protein coding region of hRif1 mRNA. 2 d after infection, 40 μg of total RNA from each cell line was hybridized with hRif1 or GAPDH probes. Relative hRif1 levels were determined by ImageQuant software using GAPDH as the loading control. (B) Depletion of hRif1 protein levels by siRNA. 50 μg of whole cell extracts from each cell line were probed with PAB2857 and α-tubulin antibodies. siRNA treatment was performed as described in A. (C) hRif1 depletion leads to cell growth inhibition in HT1080 and LOX cells. HT1080 cells and LOX cells were infected with lentiviral siRNA constructs at >95% efficiencies, as indicated by a GFP expressed from the same lentiviral vector. Cell numbers were counted every 3 d after infection and cell counts are shown as mean ± SD of plates analyzed in triplicate.

hRif1 accumulation on uncapped telomeres synthesized by hTER template mutant 47A. Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, images were viewed with the Quick Projection option. (A) hRif1 protein translocates to telomeres upon expression of hTER template mutant 47A (47A-hTer). LOX cells were infected with lentiviruses expressing wild-type hTER (WT-hTER) or 47A-hTer. 3 d after infection, cells were fixed and stained with TRF2 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). (B) hRif1 foci overlap with 53BP1 foci in LOX cells expressing 47A-hTer. Lentiviral infections were performed as described in A. Cells were stained with 53BP1 antibody (green), hRif1 antibody (red), and DAPI (blue). Bars, 10 μm.

hRif1 overexpression in S. cerevisiae perturbs telomere length maintenance dependent on the presence of the scRif1 gene. (A) Southern blot of telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Genomic DNA was isolated from yeast cells, digested with XhoI, and hybridized with a telomeric probe. (B) Histogram of bulk telomeric restriction fragments in wild-type, Δrif1, Δrif2, and Δrif1Δrif2 mutant yeast cells overexpressing hRif1 or the vector-alone control. Length of peak telomeric restriction fragments was quantified with ImageQuant from five independent sporulation experiments and is shown as mean ± SD.

Cell cycle–regulated expression and intracellular distribution of hRif1 protein. (A) hRif1 protein levels are regulated across the cell cycle. Contact-inhibited T24 cells were released into the cell cycle for 0–33 h, as indicated above the lanes. Whole cell extracts were probed with hRif1 antibody PAB2857 or α-tubulin antibody. DNA contents of synchronized cells at different time points were determined by FACS analysis. Noc., cells were released into the cell cycle for 28 h and then treated with 0.3 μg/ml nocodazole for 10 h for a M phase block. (B) hRif1 dynamically associates with chromatin during the cell cycle. LOX cells were fixed and immunostained with β-tubulin antibody (green), hRif1 antibody (red), and DAPI (blue). Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, an image of a representative single section on the z axis saved as a Photoshop file is presented. Bars, 10 μm.