Figure 2. Stable and transient depletion... | Rockefeller University Press

Figure 2.

$Figure 2. Stable and transient depletion of ATRX extends the transition to metaphase and induces congression defects. (A) HeLa-HG cells that stably express pSuper-shATRX1, pSuper-shATRX2, or pSuper (empty vector control) were generated and the level of ATRX depletion was measured by Q-RT-PCR (top) and Western blot analysis (bottom). Q-RT-PCR results were normalized to GAPDH expression and protein loading on the Western blot was controlled with α-tubulin. Error bars represent the standard deviation from triplicate samples. (B) Mitotic cells were followed in real time by videomicroscopy and the duration of prometaphase was measured for HG control and HG-shATRX1 stably depleted cells (n = 50 each) and also in transient transfections with siATRX1 (n > 120). Horizontal lines indicate the mean value of each dataset. (C) The fraction of metaphasic cells with misaligned chromosomes (inset) was evaluated in live cell cultures of HeLa-HG, HG-pSuper, HG-shATRX1, and HG-shATRX2 stable clones (n = 100 for each). (D) Live mitotic cells were followed over a 10 h period and scored for congression defects in stable (n = 50) and transient experiments (n > 120). (E) Selected panels from live cell videomicroscopy experiments of control HeLa-HG cells and ATRX-depleted cells from nuclear envelope breakdown (NEBD) to anaphase onset (AO). ATRX-depleted mitotic cells often displayed misaligned chromosomes (arrowheads) that resolved before the onset of anaphase (middle), whereas, in some cases, anaphase was initiated despite the presence of misaligned chromosomes (bottom). A subset of metaphase chromosomes underwent cycles of general decondensation and recondensation (the asterisk indicates a decondensed metaphase plate). Numbers indicate minutes after NEBD. (F) Morphological assessment of chromosome decondensation in control and transiently depleted cells. Graph depicts the percentage of metaphase spreads that display a more decondensed appearance (far right). Bars: (C) 5 μm; (E) 16 μm, (F) 20 μm.$

Stable and transient depletion of ATRX extends the transition to metaphase and induces congression defects. (A) HeLa-HG cells that stably express pSuper-shATRX1, pSuper-shATRX2, or pSuper (empty vector control) were generated and the level of ATRX depletion was measured by Q-RT-PCR (top) and Western blot analysis (bottom). Q-RT-PCR results were normalized to GAPDH expression and protein loading on the Western blot was controlled with α-tubulin. Error bars represent the standard deviation from triplicate samples. (B) Mitotic cells were followed in real time by videomicroscopy and the duration of prometaphase was measured for HG control and HG-shATRX1 stably depleted cells (n = 50 each) and also in transient transfections with siATRX1 (n > 120). Horizontal lines indicate the mean value of each dataset. (C) The fraction of metaphasic cells with misaligned chromosomes (inset) was evaluated in live cell cultures of HeLa-HG, HG-pSuper, HG-shATRX1, and HG-shATRX2 stable clones (n = 100 for each). (D) Live mitotic cells were followed over a 10 h period and scored for congression defects in stable (n = 50) and transient experiments (n > 120). (E) Selected panels from live cell videomicroscopy experiments of control HeLa-HG cells and ATRX-depleted cells from nuclear envelope breakdown (NEBD) to anaphase onset (AO). ATRX-depleted mitotic cells often displayed misaligned chromosomes (arrowheads) that resolved before the onset of anaphase (middle), whereas, in some cases, anaphase was initiated despite the presence of misaligned chromosomes (bottom). A subset of metaphase chromosomes underwent cycles of general decondensation and recondensation (the asterisk indicates a decondensed metaphase plate). Numbers indicate minutes after NEBD. (F) Morphological assessment of chromosome decondensation in control and transiently depleted cells. Graph depicts the percentage of metaphase spreads that display a more decondensed appearance (far right). Bars: (C) 5 μm; (E) 16 μm, (F) 20 μm.