Figure 6.
Loss of BLM Rescues the Increase in ALT-Related Phenotypes
(A) U2OS cells transfected with the indicated siRNAs 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.
(B) Quantification of (A). At least 100 cells per condition were counted. Data are presented as 5th–95th percentiles; n = 3; ∗∗∗∗p < 0.00001, one-way ANOVA; ns, not significant.
(C) 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.
(D) Quantification of (C). The extent of [32P] incorporation was quantified from the autoradiograph and normalized to SLX4IP+/+ siCTRL, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗∗p < 0.001 and ∗∗∗∗p < 0.00001, Student’s t test; ns, not significant.
(E) U2OS cells transfected with the indicated siRNAs were fixed, stained with propidium iodide, and analyzed using FACS. At least 10,000 cells per condition were counted.
(F) Quantification of (E). Data are presented as 5th–95th percentiles; n = 3; ∗∗∗p < 0.0001, one-way ANOVA; ns, not significant.
(G) U2OS cells were transfected with the indicated siRNAs. After 72 h of knockdown, cells were re-seeded and were then permitted to grow for 11 days before fixation and staining.
(H) Quantification of (G). The surviving fraction was normalized to SLX4IP+/+ siCTRL, which was arbitrarily assigned a value of 1. Data are represented as mean ± SD; n = 3; ∗∗∗∗p < 0.00001, Student’s t test; ns, not significant.
See also Figure S7.