TERT granules contain the mRNA encoding the pro-survival cyclin inhibitor p15INK4B.

<p><b>a)</b> TERT RNA-immunoprecipitation from cells under control or arsenite-induced stress: immunoprecipitated RNA was used for RT-PCR with specific primers for cell cycle regulators. Note that the only positive amplification product corresponds to the p15INK4B messenger, in the control but not stressed neurons (n = 2). <b>b) Upper panel.</b> p15INK4B in situ hybridization, negative control (anti-sense) and p15INK4B specific probe. Only the specific probe gives a signal, in the nucleus (DAPI positive) and in the cytoplasm. <b>Lower panel.</b> p15INK4B in situ hybridization (red) together with TERT immunofluorescence microscopy (green); nuclear labeling with DAPI (blue). Colocalization is evident in the perinuclear region (arrows in overlay image, “merge”) (n = 3). <b>c)</b> p15INK4B mRNA levels in 10 DIV hippocampal neurons in culture, under control or arsenite treatment. Arsenite does not result in degradation of the messenger (n = 3). <b>d)</b> Representative A254 gradient profile of control (Ctr) and arsenite stressed neurons (Ars); translational efficiency of p15INK4B mRNA was normalized to Histone 3 and β-actin (beta-actin) mRNA, as measured by RT-qPCR assay, using the following algorithm: 2-[ΔCt(P)- ΔCt(mRNPs)]. Stress induces p15INK4B translocation to the polysomes, reflecting higher translation. Standard errors are shown (n = 3). <b>e)</b> Western blot analysis of p15INK4B from 10 DIV hippocampal neurons in culture, in control and in neurons treated with arsenite. Tubulin is used as loading control. Note that arsenite increases the levels of p15INK4B. Bar graph on the right is the quantification of this experiment (means ± the s.d. of three different cultures; *p<0.05).</p

TERT associates to TIA1 –positive granules.

<p><b>a)</b> Western blot analysis of TERT immunoprecipitated proteins in extracts from hippocampal neurons maintained <i>in vitro</i> for 10 DIV, under basal stress conditions (control) or stressed with arsenite. Note that the two SG markers (TIA1 and P-elF2α) are precipitated whereas LSM-1, a component of PBs is not. RNase treatment does not affect TERT-TIA1 binding (n = 4). <b>b)</b> Western blot analysis of TERT immunoprecipitated proteins in extracts from old mice. As in the <i>in vitro</i> experiments, TIA1 and P-elF2α are precipitated whereas LSM-1 is not (n = 3). <b>c)</b> Western blot analysis of TIA1 immunoprecipitated proteins in extracts from hippocampal neurons maintained <i>in vitro</i> for 10 DIV, under basal stress conditions (control) or stressed with arsenite. Again, RNase treatment does not affect TERT-TIA1 binding (n = 4). <b>d)</b> Western blot analysis of TIA1 immunoprecipitated proteins in extracts from old mice. Note that TIA1 and P-elF2α are precipitated whereas LSM-1 is not (n = 3). <b>e)</b> The known TIA1 target ß-actin mRNA is amplified in RNA purified from TERT immunoprecipitate, whereas another known TIA1 target, Caspase-7, is not. The first lane corresponds to the markers (n = 2). <b>f)</b> Confocal microscopy images of neurons double labeled TERT (green)-TIA1 (red) (upper row) and TERT (green)-PABP (red) (lower row). Numerous foci of colocalization exist (quantified in the bar graph: mean ± the s.d. from three different experiments). Scale bar: 10 µm. <b>g)</b> Confocal microscopy images of neurons infected with scrambled or shTERT, stained for TERT (red) and counterstained for TIA1 (blue). The reduction in TIA1 labeling is not significant (mean ± the s.d. from three different experiments). Scale bar: 10 µm.</p

TERT exerts its anti-apoptotic role through regulation of p15INK4B messenger,

<p>a<b>)</b> Western blot analysis of TERT and p15INK4B levels in 10 DIV hippocampal neurons infected with scrambled or shTERT; Tubulin is used as loading control. Note that p15INK4B levels are reduced by TERT downregulation (n = 2). <b>b)</b> Western blot analysis of p15INK4B levels in control 10 DIV hippocampal neurons and infected with p15INK4B shRNA. The reduction in protein content is more than 50% (bar graph on the right, n = 2). <b>c)</b> Tunel assay of 10 DIV hippocampal neurons under control conditions (control) and after infection with an empty vector (vector) or with the shRNA for p15INK4B (shp15INK4B). The bar graph illustrates the significant cell death under this last condition (mean ± the s.d. of three different cultures; * p<0.05).</p