Figure 2.
Loss of SLX4IP in ALT-Positive Cells Increases ALT-Related Phenotypes
(A) Genomic DNA was isolated from U2OS cells and processed to detect Phi29-dependent telomere circles. The Phi29 amplification products were detected by Southern blotting using a γ[32P]-labeled telomeric (TTAGGG) probe.
(B) Quantification of (A). The extent of [32P] incorporation was quantified from the autoradiograph and normalized to SLX4IP+/+, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗p < 0.01, Student’s t test.
(C) U2OS cells were fixed and processed for PML immunofluorescence followed by telomeric PNA (TelG) FISH. Scale bar represents 10 μm. Dashed lines indicate nucleus outlines (as determined using DAPI staining; not shown). Insets represent 3× magnifications of the indicated fields.
(D) Quantification of (C). At least 100 cells per condition were counted. Data are presented as 5th–95th percentiles; n = 3; ∗∗∗∗p < 0.00001, Student’s t test.
(E) U2OS cells were fixed, and metaphases were processed for chromosome-orientation FISH using PNA probes against the C-rich (TelC) and the G-rich (TelG) telomere strand. Scale bar represents 100 μm.
(F) Quantification of (E). At least 25 metaphases per condition were counted. Data are presented as 5th–95th percentiles; n = 3; ∗∗∗∗p < 0.00001, Student’s t test.
(G) U2OS cells were fixed and processed for γ-H2AX immunofluorescence followed by telomeric PNA (TelG) FISH. Scale bar represents 10 μm. Dashed lines indicate nucleus outlines (as determined using DAPI staining; not shown). Insets represent 3× magnifications of the indicated fields.
(H) Quantification of (C). At least 100 cells per condition were counted. Data are presented as 5th–95th percentiles; n = 3; ∗∗∗p < 0.0001 and ∗∗∗∗p < 0.00001, Student’s t test.
(I) U2OS cells were fixed, and metaphases were processed for telomere PNA (TelG) FISH. Scale bar represents 100 μm.
(J) Quantification of (H), showing the telomere fluorescence distribution of individual telomere dots. At least 25 metaphases per condition were counted. Mean fluorescence is indicated by the red horizontal line; shown is a representative experiment; ∗∗∗∗p < 0.00001, Student’s t test.
See also Figure S4.