PMC

IF analysis of the nuclear localization of SKIIP and TRF2 in directly fixed (A) or pre-extracted (B) nuclei. Co-localization events in pre-extracted nuclei have been indicated by arrows.

(A) Schematic overview of the screen layout. (B) Primary data of all siRNA pools. The positive control (TRF2 OTP siRNA pool), SKIIP and the TRF1 pseudogene LOC283523 are indicated.

IF-FISH images after the suppression of LOC283523 and SKIIP using the siGENOME pools. RLUC-siRNA transcfected cells serve as control. Telomeres were detected by hybridization to a FITC coupled TTAGGG probe and damage foci were visualized by 53BP1 staining.

(A) Western analysis of SKIIP and TRF2 72 hours post transfection with OTP siRNA pools. gamma-Tubulin serves as loading control. All knock-downs achieved greater than 95% suppression of the targets. (B) Visualization of telomeric and non telomeric DNA damage foci in cells depleted for SKIIP using OTP siRNA pools. RLUC-siRNA transcfected cells serve as control. Telomeres were detected by hybridization to a FITC coupled TTAGGG probe and damage foci were visualized by 53BP1 staining.

(A) Western analysis of TRF1 and TRF2 expression post transfection with OTP siRNA pools at the indicated time points post transfection. gamma-Tubulin serves as loading control. All knock-downs achieved greater than 80% suppression of the targets. (B) IF-FISH based TIF analysis in cells depleted for TRF1 or TRF2. 53BP1 staining was used as marker for DNA damage and a FITC coupled telomeric FISH probe was used to detect telomeric repeats. Untr: untransfected control, RLUC: control siRNA targeting Renilla luciferase, siGLO: cells transfected with siGLO green transfection indicator, ch: media was changed 24 hours post transfection, IR: cells subjected to 10 Gray of ionizing radiation.