## 3 Results
### The Cerebellar Transcriptome Profile of ATM-Null Mice at 12 Months of Age
In parallel to the desired reduction of _ATM_ in SH-SYSY knockdown cells documented in Figure 4a, these further studies (see Figure 5 and Figure S4) confirmed strong genotype-dependent downregulations for _ATMIN_ mRNA (to 71% and 76%) as a mediator of ATM responses to osmotic and oxidative stress. _NR4A1_ and _FOXO3_ mRNA, as immediate-early mediators of phosphorylation signals to the nucleus, were both found to be responsive to CQ-stressor treatment (1.5-fold and 1.6-fold increase, respectively), while displaying abrogated induction in shATM cells (Figure 5a, Figure S4a). In addition, the CQ-triggered inductions of calcium-dependent kinases _CAMK2A_ and _CAMK4_ mRNA (4.1-fold and 1.3-fold, respectively) were significantly impaired upon _ATM_-KD (Figure S4a). Importantly, an ATM-dependent mRNA downregulation (to 32% and 37% for unstressed and stressed conditions, respectively) was also observed for the ataxia gene _ITPRI_ and might therefore be interpreted as a loss-of-function that may have a primary role in the pathogenesis of autosomal recessive A-T, while the other ataxia genes _GRID2_[99; 100] and _MME_[101] showed ATM-dependent mRNA upregulation (2.4-fold and 3.4-fold for _GRID2_; 1.6-fold and 3.0-fold for _MME_, in unstressed and stressed _ATM_-KD cells) that may represent compensatory efforts, and the ataxia transcript _RORA_[102; 103] exhibited only a response to osmotic stress (1.8-fold increase in CQ treated cells; Figure 5a). As further evidence for compensatory reactions to osmotic stress, _RRAGD_ mRNA (encoding Ras-related RagD amino acid sensor [104; 105]) showed significant upregulation after CQ treatment, and even bigger upregulation after _ATM_-KD (4.0-fold induction in NT CTRL cells and 8.1-fold increase in stressed shATM cells, Figure 5a). In addition, _SGKI_ transcript induced only upon CQ treatment in shATM cells (1.4-fold, \(p\) = 0.3903 and 2.4-fold, \(p\) = 0.0007) corroborates the presence of osmotic stress (Figure S4a). As a putative modifier of vasodilitation, _ECEL1_ mRNA [106; 107] was found upregulated after CQ stress in NT CTRL cells (1.4-fold, Figure 5a) and after oxidative stress in _ATM_-KD cells (1.4-fold), as well as after CQ stress during ATM kinase inhibition (1.2-fold) (Figure S4b). Figure 4: _ATM_-KD successfully models the dysregulations found in vivo, while inhibition of ATM kinase function is not effective. (**a**) Scheme of chloroquine (CQ) stressor treatment in _ATM_-KD  
SH-SY5Y cells (upper panel) and corresponding _ATM_ transcript levels determined via RT-qPCR (n = 3, lower panel). **(b)** Scheme of CQ stressor treatment in parental SH-SY5Y cells with KU-55933 ATM kinase inhibitor pretreatment (upper panel) and corresponding _ATM_ transcript levels determined via RT-qPCR (n = 3, lower panel). **(c)** Comparison of ATM interactor _USP_2 mRNA and **(d)** immediate early transcript _PER1_ levels in _ATM_-KD (left) vs. ATM-kinase-inhibited parental cells (right) after CQ stress, as determined via RT-qPCR (n = 3). **(e)** Reduction of _USP_2 transcripts upon CQ-stress and in _ATM_-KD was largely reproduced by LY-294002 stressor treatment, but not after BLEO and NaARS treatment of _ATM_-KD cells (n = 3). **(f)** BLEO, NaARS and LY-294002 stressor treatments did not reproduce the ATM dependent _PER1_ transcript induction present in aged ATM-null cerebellum, which was however successfully modelled after CQ-stressor treatment of _ATM_-KD cells (n = 3) as seen in panel **(d)**. **(g)** PER1 protein was reduced by CQ-stress administration in NT CTRL cells and generally in shATM cells, as determined by quantitative immunoblots (n = 3). For statistical trends, the precise _p_-value was shown. Asterisks display significance. * = _p_\(\leq\) 0.05, *** = _p_\(\leq\) 0.01, *** = _p_\(\leq\) 0.001, *** = _p_\(\leq\) 0.0001, ns = non-significant. Data are displayed as bar plots with data points, mean \(\pm\) SEM. Quantitative immunoblots were conducted when commercial antibodies were available to us with sufficient specificity and sensitivity to detect the endogenous protein levels.