1. Volume 20, Issue 4, Pages 868-880 (July 2017)
Aberrant Proteostasis of BMAL1 Underlies Circadian Abnormalities in a Paradigmatic mTOR-opathy 
Jonathan O. Lipton, Lara M. Boyle, Elizabeth D. Yuan, Kevin J. Hochstrasser, Fortunate F. Chifamba, Ashwin Nathan, Peter T. Tsai, Fred Davis, Mustafa Sahin 
Cell Reports 
Volume 20, Issue 4, Pages (July 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 868-880DOI: (10.1016/j.celrep.2017.07.008)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Abnormal Circadian Rhythms in Brain-Specific Tsc1 Knockout Mice
(A) Outline of the experimental protocol for measurement of core body temperature in Tsc1 brain-specific knockout mice.
(B) Daily core body temperature measurements in representative SynICre;Tsc1flox/flox mice compared with controls. Note that the daily oscillations in temperature in DD are dysregulated in the mutant compared with controls.
(C) Representative mean temperature recordings as a function of circadian time under L/D or D/D conditions for mice of the indicated genotype.
(D) Fast Fourier analysis demonstrating amplitude of power spectrum relative to period (1/frequency).
(E) Histogram of the circadian period under DD conditions in SynICre;Tsc1flox/flox mice compared with controls. Student’s t test, ∗p < 0.05, n = 8/genotype.
(F) Histogram of mean circadian amplitude from the indicated genotypes. Student’s t test, ∗∗p < 0.01.
(G) The mean core body temperature (Tcb) does not differ between SynICre;Tsc1flox/flox mice and controls.
(H) Histogram of diurnal variation of Rev-Erbα in the cortex of SynICre;Tsc1flox/flox mice. n = 3 biological replicates, each measured in duplicate; Student’s t test; ∗∗p < 0.01.
All error bars indicate ± SEM. See also Figures S1 and S2.
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4. Figure 2
The Shortened Free-Running Period in Tsc2+/− Mice Is Responsive to Rapamycin
(A) Representative double-plotted actograms demonstrating wheel-running activity in Tsc2+/− and Tsc2+/+ mice. Shading demonstrates the timing of light-dark periods.
(B) Circadian period under DD conditions (error bars reflect minima and maxima of n = 9/genotype), Student’s t test, ∗∗p <
(C) Fast Fourier transform power under DD conditions (error bars reflect minima and maxima of n = 9/genotype). Student’s t test; ns, not significant.
(D) Activity quantified as number of wheel rotations per minute under DD conditions (error bars reflect minima and maxima of n = 9/genotype); Student’s t test.
(E) Representative double-plotted actograms demonstrating wheel-running activity in Tsc2+/− and Tsc2+/+ mice. Shading demonstrates the timing of light-dark periods.
(F) Circadian period under DD conditions before and after rapamycin injection (error bars reflect minima and maxima of n = 9/genotype without rapamycin, n = 3/genotype with rapamycin),; two-way ANOVA, ∗p < 0.05, ∗∗p = 0.02.
All error bars indicate ± SEM. See also Figure S1.
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5. Figure 3 Abnormal Circadian Timing in Tsc2 Knockout Cells
(A) Quantitative PCR of the indicated core clock components in unsynchronized MEFs (Tsc2−/− compared with Tsc2+/+ cells, n = 3 biological replicates/genotype run in duplicate and normalized to wild-type cells. Student’s t test, ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(B) qPCR of the indicated core clock components in dexamethasone-synchronized Tsc2−/− compared with Tsc2+/+ cells demonstrates abnormal circadian timing of relative mRNA expression. Two-way ANOVA (zeitgeber time [ZT] and genotype) with Bonferroni post hoc test of every data point compared with Tsc2+/+ cells at ZT0, normalized arbitrarily to wild-type cells at ZT0. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < , n = 3 biological replicates/genotype, each run in duplicate.
All error bars indicate ± SEM.
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6. Figure 4
Increased BMAL1 Protein Expression in Tsc-Deficient Cells and Animals
(A) Western blot of whole-cell MEF lysates of the indicated genotype; n = 6 biological replicates, Student’s t test, ∗∗∗p <
(B) Immunocytochemistry of Tsc2 MEFs of the indicated genotype serum-starved overnight or maintained in serum-containing medium. Quantification of nuclear/cytoplasmic ratio was normalized to serum-starved wild-type cells. Scale bar, 50 μm. ∗∗∗p < , one-way ANOVA with Bonferoni post hoc test.
(C) Representative western blot of nuclear and cytoplasmic fractions of Tsc2 MEFs of the indicated genotype treated as shown (n = 3 replicates). Nuclear and cytoplasmic fractions were run simultaneously and membranes were bisected prior to antibody application, incubated and processed together and then re-assembled for comparison.
(D) Western blots from brain lysates from Tsc2+/− and Tsc2+/+ littermates collected at the indicated circadian times under constant conditions. Right: densitometry measurements of BMAL1 relative to actin normalized to Tsc2+/+ controls at CT3. Two-way ANOVA, p = 0.02 for interaction, p = 0.02 for genotype, p = 0.02 for circadian time.
(E) Representative western blots from SynICre;Tsc1flox/flox brains compared with controls with densitometry measurements of BMAL1 relative to tubulin; n = 4/genotype, Students t test, ∗∗∗p <
(F) Representative BMAL1 immunohistochemistry of suprachiasmatic nuclei at ZT2 from the indicated genotypes.
All error bars indicate ± SEM. See also Figure S3.
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7. Figure 5 BMAL1 Translation Is Increased in Tsc2−/− MEFs
(A) Experimental outline of BONCAT and affinity purification of de novo synthesized proteins.
(B) Representative immunoblots of cytoplasmic lysates and streptavidin affinity purification of biotinylated L-AHA-tagged de novo synthesized protein demonstrates increased BMAL1 in Tsc2−/− cells. See also Figure S4.
(C) Quantification of total BMAL1 and de novo synthesized BMAL1 in MEFs of the indicated genotype; n = 3 biological replicates, Student’s t test, ∗∗p <
(D) Tsc2 MEFs of the indicated genotype were pulse-labeled with 35S-Met/Cys for 1 hr, followed by immunoprecipitation with BMAL1 monoclonal antibody and quantification by densitometry. See also Figure S5.
All error bars indicate ± SEM.
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8. Figure 6
Impaired BMAL1 Ubiquitination, Degradation, and UBE3A Association in Tsc2−/− Cells
(A) Tsc2 MEFs were treated with 100 μg/mL cycloheximide, and whole-cell lysates were collected at the indicated times for a western blot.
(B) Quantification of the experiments in (A), analyzed by two-way ANOVA for genotype and time, with Bonferoni correction; ∗∗p < 0.01.
(C) Luminometry of the CMV-BMAL1-Luciferase fusion reporter transfected into either Tsc2+/+ or Tsc2−/− MEFs and incubated with cycloheximide. Shown is the average luminescence ± SEM of 6 samples/genotype/time point normalized to time 0 for each genotype. Also shown is a histogram of the exponential decay rate constant (1/hr) k ± SD, ∗∗∗p < , t test.
(D) Tsc2 MEFs were treated with 100 nM bortezomib and either DMSO or 250 nM Torin 1 for 4 hr, followed by cell lysis and anti-BMAL1 immunoprecipitation. ∗, IgG heavy chain; black arrow, presumed low-molecular-weight ubiquitinated BMAL1. All images represent samples run on the same membranes cut for visual clarity.
(E) Model of mTOR regulation of BMAL1 proteostasis in the presence or absence of TSC1/2 function.
All error bars indicate ± SEM.
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9. Figure 7
Tsc-Deficient Cells and Mice Demonstrate a Rapid Responses to Phase Shifts Ameliorated by Lowering the Bmal1 Dose
(A) Per1 expression during serum synchronization in Tsc2−/− and control MEFs treated with or without 250 nM Torin 1; n = 3 biological replicates/genotype, and each sample was run in duplicate; two-way ANOVA (time and genotype) with Bonferroni correction; ∗∗∗p <
(B) Detrended bioluminescence of U2-OS(Bmal1:luciferase) reporter cells treated with DMSO, 100 μM rapamycin, or 250 nM Torin 1 after dexamethasone synchronization.
(C) Quantification of time to first nadir in Bmal1:luciferase in U2-OS(Bmal1:luciferase) cells; n = 6 from two independent experiments; one-way ANOVA, Bonferroni post-test, ∗∗p < 0.01, ∗∗∗p <
(D) Representative actograms of Tsc2+/+, Tsc2+/−, and Tsc2+/−;Bmal1+/− littermates under LD conditions after 6-hr phase advancement.
(E) Representative western blot of cortical lysates separated into cytoplasmic and nuclear fractions from the indicated genotypes (n = 3/genotype).
(F) Phase resetting of the indicated genotypes quantified as time to reach the new phase; the number of animals per genotype is noted in parenthesis (two-way ANOVA with Bonferroni post-tests, p = for interaction, p < for genotype, p < for time; for individual time point comparisons, ∗p < 0.05 and ∗∗∗p < 0.001).
(G) Free running period under DD conditions for the indicated genotype (one-way ANOVA, ∗∗p < 0.01).
All error bars indicate ± SEM.
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