LC-MS/MS plasma sample DIA settings.

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Altered brain proteins at FDR<15% in Active versus Control participants.

A-K) Expression of the 11 altered brain proteins at FDRFig 3A. Error bars indicate SEM. L) The 11 altered proteins were associated with 2 signaling pathways having a p value of overlap 2. The same four proteins (FGA, FGB, FGG, PLG) were associated with activation of the coagulation system (p value of overlap = 1.45 x 10−9, z = 2.00) and acute phase response signaling (p value of overlap = 1.23 x 10−6, z = 2.00). (TIF)</p

Proteomics in whole brain homogenate.

Proteomics was performed in surgically resected brain tissue from Active and Control participants. A) PCA in brain tissue indicated no segregation of Active and Control participants in PCA1 (p = 0.73) and with some segregation in PCA2 (p = 0.016). Clinical diagnoses (FCD, TSC) are noted as well. B) Differential expression analysis in brain tissue indicated that there were 11 significantly altered proteins in Active versus Control participants at an FDR S1 Table and S1 Fig. There were no significant proteins at FDR C) WGCNA of brain proteomics and phospho-S6 evaluated in total brain homogenate by western blot, regardless of everolimus and clinical diagnoses. There were 1109 proteins that correlated with phospho-S6 levels (P235/236, p −6, R2 = 0.87) in the M-Brown module. D) The top significantly correlated protein with P240/244 was a negative correlation with ANK2 (p = 7.92 x 10−5, R2 = 0.74) in the M-Brown module. E) Module trait correlation identified 5 significantly associated modules with phospho-S6 (p < 0.05). Modules were clustered by eigenprotein adjacency (relatedness to other modules) on the left. Name of module is indicated by “M-color.” P values are indicated for those modules with p < 0.05 correlation. Positive correlation is indicated in red and negative correlation in blue. Top module GO annotations are noted on the right (FDR < 5%, at least 5 proteins/annotation). Several modules did not have a significant GO annotation (“n.s.”).</p

Metabolomics raw data in brain.

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LC-MS/MS proteomics in brain.

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Ribosomal S6 phosphorylation in whole brain homogenate.

A) Representative western blot images from surgically resected brain tissue of Active participants after taking 4.5 mg/m2 everolimus for 7 days and controls. Total S6, phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), and beta-actin as loading control were evaluated in 14 participants. TSC participants are indicated “*”, while all others are FCD participants. B) Quantification of total S6 relative to actin indicates variability in baseline levels from sampled brain tissue, as well as one patient with increased expression of a smaller detected band (NYU-002). C) Percentage of phospho-S6 (Ser235/236) relative to total S6 indicates an average 1.19-fold decrease when comparing all Active participants to controls (p = 0.67). The highest level was seen in NYU-004. D) Percentage of phospho-S6 (Ser240/244) relative to total S6 indicates an average 1.15-fold decrease when comparing all Active participants to controls (p = 0.66). The highest level was seen in NYU-004. Error bars indicate SEM.</p

LC-MS/MS brain IPA pathways.

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Whole western blot images for phospho-S6 (Ser235/236).

A) All cases (n = 14) were evaluated by western blot for phospho-S6 (Ser235/236) quantification on 2 blots, as depicted in Fig 1A. Bands were visualized after ECL on a BioRad ChemiDoc. Quantification was performed on bands in the red outlined box in Fiji ImageJ. One sample (NYUC001) was included on both gels to allow for normalization across blots for all samples. A’) Corresponding ladder for panel A is shown in the colorometric brightfield image. B) On the same blots, actin was evaluated after stripping the phospho-S6 (Ser235/236). B’) Corresponding ladder for panel B is shown in the colorometric brightfield image. C) On the same blots, total S6 was evaluated with no stripping (both actin and total S6 are present). C’) Corresponding ladder for panel C is shown in the colorometric brightfield image. (PDF)</p

Metabolomics in whole brain homogenate.

Global polar metabolomics by LC-MS was performed on surgically resected brain tissue from Active and Control participants. A) PCA indicated distribution of participants, as well as clinical diagnoses (FCD, TSC). There was no segregation of Active and Control participants in PCA1 (p = 0.32) or PCA2 (p = 0.48). B) Differential expression analysis (p 2 (fold change) > 1) identified 14 metabolite features that were increased in Active participants, associated with the indicated annotations detailed in C).</p

Histological quantification of ribosomal S6 phosphorylation in gray matter.

Representative images show total S6, phospho-S6 (Ser235/236), and phospho-S6 (Ser240/244) (red) in resected cortical tissue from FCD participants that were Controls A-C) or Active D-F) and in TSC participants that were Controls G-I) or Active J-L). M) Quantification of total S6 expression in the gray matter indicated no difference in baseline expression between Active and Control participants. N) Quantification of percent phospho-S6 (Ser235/236) relative to total S6 in the gray matter indicated an average 1.56-fold decrease when comparing all Active participants to Controls (p = 0.37). O) Quantification of percent phospho-S6 (Ser240/244) relative to total S6 in the gray matter indicated an average 5.55-fold decrease when comparing all Active participants to Controls (p = 0.22). Histology was performed on all cases with available FFPE, excluding 1 Active FCD case. Error bars indicate SEM. Scale bar represents 100 μm.</p