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ana23943-sup-0001-suppinfo01.tif17687KSuppl. Figure 1: Acute loss of Tsc1 leads to increased TORC1 activation in the cortex, but not in the cerebellum. Representative Western blots and quantification of TSC1, TSC2 and S6 S235/236 phosphorylation in cortical (A) and cerebellar (B) lysates of Tsc1f/–::CreERT+ mice sacrificed two or four days (2d or 4d) after the first tamoxifen injection or two days after injection of vehicle only. Tamoxifen-injected Tsc1f/f mice without CreERT (control) are shown for comparison. We used 3 mice for every group: Tsc1f/– (vehicle); Tsc1f/– (2d) and Tsc1f/– (4d); protein levels were normalized to the levels of tamoxifen injected control mice (control; N=3). Error bars represent the SEM. C, Representative Western blot comparing S6 S235/236 phosphorylation in cerebellar, hippocampal and cortical lysates of tamoxifen-injected control and Tsc1f/– mice sacrificed four days (4d) after initiation of gene deletion. For quantification values were normalized to hippocampus control. Error bars represent the SEM.
ana23943-sup-0002-suppinfo02.tif4002KSuppl. Figure 2: Phosphorylation of 4EBP1 is unchanged after acute deletion of the Tsc1 gene. Quantification of Western blots. Tsc1f/– (2d): N=3, Tsc1f/– (4d): N=3; levels of the 3 different phosphorylated isoforms of 4EBP1 (α,δ,γ) were normalized to the total (sum of all three forms). Error bars represent the SEM.
ana23943-sup-0003-suppinfo03.tif31925KSuppl. Figure 3: Acute loss of Tsc1 increases TORC1 activity in cortex and hippocampus. A Microscopic images (5x magnification) of different brain regions of tamoxifen treated Tsc1f/f mutants with and without CreERT stained for phosphorylation of S6 (pS6 Ser235/236) four days after initiation of gene deletion. pS6 is strongly increased in cortex and hippocampus, but less in striatum and thalamus. High levels of pS6 in cerebellar Purkinje cells are already present before onset of gene deletion. B Example of an enlarged pS6 positive cell in the cortex of tamoxifen treated Tsc1f/f CreERT+ mutant (40x magnification).
ana23943-sup-0004-suppinfo04.tif36419KSuppl. Figure 4: Rheb1 heterozygosity decreases TORC1 activity and delays seizure onset in Tsc1f/f mice. A, Phosphorylated S6 (Ser235/236) is significantly increased in tamoxifen induced Tsc1f/f Rheb+/+ mice compared to vehicle treated control mice (Tsc1f/f Rheb+/+) but not in tamoxifen induced Tsc1f/f Rheb+/- mice (One-way ANOVA, Bonferroni post-hoc test; Tsc1f/f Rheb+/+ tamoxifen versus control p<0.05; Tsc1f/f Rheb+/+ vehicle versus Tsc1f/f Rheb+/- tamoxifen: p=0.37; Tsc1f/f Rheb+/+ tamoxifen versus Tsc1f/f Rheb+/- p=0.14). Western blots were performed on hippocampal lysates from mice sacrificed 5 days after the initiation of gene deletion. Values were normalized to the control mice. Error bars represent the SEM. B, top graph: Seizure onset shows a shift from a median of 8 days in Tsc1f/f Rheb+/+ mice to a median of 11.5 days in Tsc1f/f Rheb+/- mice (Log-rank (Mantel-Cox) test: p=0.16; N=6 per group). Bottom graph: the survival of the Tsc1f/f Rheb+/- mice is slightly increased compared to Tsc1f/f Rheb+/+ (median survival: 15.5 days vs. 11 days; Log-rank (Mantel-Cox) test: p=0.10; N=4 per group)
ana23943-sup-0005-suppinfo05.tif12287KSuppl. Figure 5: Low dose rapamycin treatment (5 mg/kg) reduces seizure frequency in Tsc1f/f mice. Tsc1f/f mice were treated with 5 mg/kg rapamycin after seizure onset (varying between 5–7 days after initiation of gene deletion). Rapamycin reduced seizure frequency, but did not prevent the death of the mice 2–4 days after the onset of epilepsy and 7–9 days after the initiation of gene deletion. N=4; error bars represent the SEM.
ana23943-sup-0006-suppinfo06.tif11199KSuppl. Figure 6: Rapamycin is still detectable in the brain, but not in the blood, 16 days after cessation of treatment. Tsc1f/f without CreERT were treated with 10 mg/kg rapamycin for 4 consecutive days and followed by three more injections every other day. Mice were sacrificed 1, 7 or 16 days after the last rapamycin injection and blood and brain tissue was collected to measure rapamycin levels. Mean values with SEM are shown (N=4 per time point) and an exponential decay equation (one phase) was fitted to the data (R2: blood: 0.99; brain: 0.96; decay constant: blood: 26.4 hour-1; brain; 3.4 hour-1).
ana23943-sup-0007-suppinfo07.tif15128KSuppl. Table: Passive membrane properties and firing pattern of pyramidal neurons in the CA1 cell layer of hippocampal slices. Pyramidal neurons from tamoxifen treated Tsc1f/ and Tsc1f/ control mice without CreERT mice were measured 4 days after the first tamoxifen injection, before the mice developed seizures. Average values with the SEM are shown (Tsc1f/-::CreERT+: N=24; Tsc1f/- ::CreERT: N=40).

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