Fig 2: Characterization of dysmorphic astrocytes. (A) Hematein-eosin staining of a cytomegalic astrocyte (arrowhead) in a white matter nodule displaying a long thick extension. Also note the presence of numerous SF cells. (B) GFAP immunostaining of astrocytes (arrowheads) and Giant cell (*). (C) Glutamine synthetase staining of a Giant cell (*) and a dysplastic astrocyte (arrowhead) in a tuber. Note that astrocytic extensions were thicker and longer than that of Giant cells. (D) Vimentin immunostaining of a dysmorphic cytomegalic astrocyte in a white matter nodule. (E) Filamin A immunostaining of an astrocyte (arrow head) and some Giant cells (*) in the superficial white matter. (F) Immunostaining with RPS6 R232H of glial cells in a subependymal nodule. Arrowhead points to a dysmorphic astrocyte. Pictures were taken from 25GW (B), 30 (F), 34GW (A) and 36GW (C–E); from white matter nodules (C–E) and subependymal nodules (A,B,F). Scale bars: 10 µm. The frieze schematically represents the intensity of immunostaining of tuberous lesions throughout development.

Fig 3: Bioinformatics analysis. A: K-M survival analysis of RPS6 (S6) mRNA expression. B: K-M survival analysis of PDCD1 (PD1) mRNA expression. C: K-M survival analysis of CD274 (PD-L1) mRNA expression.

Fig 4: Working model representing the proposed signaling pathways induced by vitamin D signaling in regulating skeletal muscle mass. Active vitamin D bound to vitamin D receptors maintains proteostasis by inhibiting p-Stat3 signaling and downstream C/EBP d activity, thereby inhibiting the production of Myostatin. In fast muscles, active vitamin D additionally promotes p-Stat5 expression that could function to inhibit Stat3 expression and function. In the absence of vitamin D signaling, increased levels of Myostatin induces p-Smad3 signaling, which in turn inhibits components of the mTOR pathway, p-p70S6 kinase, and p-rpS6. Translocation of active FOXO3 into the nucleus due to reduced mTOR pathway signaling can upregulate the expression of various atrogenes. Paradoxically, autophagy regulators and targets of FOXO3 signaling, LC3b and Bnip3, display decreased expression, suggesting a block in the autophagic process and possibly an inhibition of recruitment of FOXO3 to the promoters of these genes. Additionally, active vitamin D reduces IL-6 production in slow muscles, possibly through the inhibition of the NF-kb complex that further attenuates p-Stat3 signaling

Fig 5: VDR-/- muscles are characterized by an increase in atrophic gene expression, a decline in mTOR pathway components, and a block in autophagic gene expression. a The soleus and TA muscles from 6-week-old WT and V mice were assessed for levels of Myostatin transcript by qRT-PCR. Myostatin transcript levels in the soleus and TA muscles of V mice are normalized to those in WT (n = 6) (*p < 0.05). Myostatin is upregulated in the soleus and TA muscles of V compared to WT. b Graph showing the quantitation of western blot analysis of lysates from dissected soleus muscles (Fig. 1b) and TA muscles (Fig. 1c) from WT and V mice at 6 weeks of age probed for p-Smad3 antibody (n = 5) (**p < 0.01, ***p < 0.005). GAPDH serves as a loading control. c–f Representative western blot analysis of lysates from dissected soleus muscles (Fig. 1b) and TA muscles (Fig. 1c) from WT and V mice at 6 weeks of age probed for p-p70S6K antibody and S6K antibody (c, d) and p-rpS6 antibody and total rpS6 (e, f). Graphs below each blot show ratios of p-p70S6K to total S6K and p-rpS6 to total rpS6, respectively (n = 5) (**p < 0.01, ***p < 0.005). GAPDH serves as a loading control. g, h Soleus and TA muscles from 6-week-old WT and TA muscles were assessed for known markers of atrophy, Foxo3, Murf1, and C/EBP d transcripts by qRT-PCR. While C/EBP d was upregulated in both muscles sets, Foxo3 and Murf1 showed differential fiber-type-specific expression (n = 5) (**p < 0.01, ***p < 0.005). i–j Soleus and TA muscles from 6-week-old WT and TA muscles were assessed for known markers of autophagy, LC3b and Bnip3 transcripts, by qRT-PCR. Both transcripts were downregulated in V muscles compared to WT muscles. All transcript levels in V are normalized to those in WT (n = 5) (**p < 0.01, ***p < 0.005)