Fig 2: 400 Hz-hfrTMS leads to increases the co-activated rpS6-positive and Arc, c-fos protein-positive neurons in the motor cortex. (A–C) Pattern of immunostaining for rpS6 in sham control animal (A). Pattern of immunostaining for Arc (B) and c-fos (C) in sham control animal. (D–F) Pattern of immunostaining for rpS6 (D), Arc (E) and c-fos (F) 30 min, after 400 Hz hfrTMS. Note that many neurons including some pyramidal neurons in layer V were strongly positive for rpS6 and Arc protein, the larger pyramidal neurons in layer V were less intensely stained for Arc and that the distribution of c-fos did not align with the distribution of Arc-positive pyramidal neurons within the layer V.

Fig 3: Induction of rpS6 phosphorylation depends on NMDA receptor activation. (A) Evoked responses recorded with a saline-filled microelectrode. (B) Evoked responses recorded with a bicuculline-filled microelectrode. (C) Evoked responses recorded with saline-microelectrode. (D) Evoked responses recorded with microelectrode filled with bicuculline and APV combined. (E) Immunostaining for p-ser235/236 in a section ipsilateral to test pulse stimulation in the presence of bicuculline. (F) High-magnification image of E. (G) Immunostaining for p-ser235/236 in a section contralateral to test pulse stimulation. (H) High-magnification image of G. (I) Immunostaining for p-ser240/244 in a section ipsilateral to test pulse stimulation in the presence of bicuculline. (J) High-magnification image of I. (K) Immunostaining for p-ser240/244 in a section contralateral to test pulse stimulation. (L) High-magnification image of K. (M) Immunostaining for p-ser235/236 in a section ipsilateral to test pulse stimulation in the presence of bicuculline and APV combined. (N) High-magnification image of M. (O) Immunostaining for p-ser235/236 in a section contralateral to the test pulse stimulation. (P) High-magnification of O. (Q) Immunostaining for p-ser240/244 in a section ipsilateral to test pulse stimulation in the presence of bicuculline and APV combined. (R) High-magnification image of Q. (S) Immunostaining for p-ser240/244 in a section contralateral to the test pulse stimulation. (T) High-magnification image of S. Scale bars: G, 200 µm; H, 50 µm.

Fig 4: Local infusion of the MEK inhibitor, U0126, attenuates dendritic phosphorylation of rpS6. (A) Immunostaining of phospho-ERK in a section within the injection site. (B) High-magnification image A at the site of infusion. (C) Immunostaining of phospho-ERK in a section distant from the injection site. (D) High-magnification image of C. (E) Immunostaining for p-ser235/236 ipsilateral to stimulation in a section distant from the injection site. (F) High-magnification image of E. (G) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the injection site, ipsilateral sections within the infusion area, and contralateral, nonstimulated sections. (H) Statistical assessment at four sites along the dentate gyrus, GCL, IML, MML, OML (ser235/236, N = 7), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction (F(6,72) = 13.02, P < 0.0001), a significant main effect of treatment (F(2,72) = 121.41, P < 0.0001), and a significant main effect of region (F(3,72) = 94.30, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed a significant difference between distant sections and contralateral sections at all measuring sites. There was also a significant difference between sections within the infusion area and contralateral sections in the GCL, IML, and MML. Notably, there was a significant difference between distant sections and sections within the infusion area in the IML, MML, and OML (two-way ANOVA, P < 0.05). (I) Immunostaining of p-ser235/236 ipsilateral to the stimulation in a section within the injection site in the presence of U0126. (J) High-magnification image of I. Note local infusion of U0126 reduces phosphorylation of rpS6 throughout the molecular layer of the dentate gyrus. The distinct band of rpS6 phosphorylation is not discernable. (K) Immunostaining of p-ser235/236 contralateral to the stimulation. (L) High-magnification image of K. (M) Immunostaining of p-ser240/244 in a section distant from the injection site. (N) High-magnification image of M. (O) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the injection site, ipsilateral sections within the injection site, and contralateral sections. (P) Statistical assessment at four sites along the dentate gyrus, GCL, IML, MML, OML (ser240/244, N = 7), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction (F(6,72) = 3.89, P = 0.0020), a significant main effect of treatment (F(2,72) = 83.75, P < 0.0001), and a significant main effect of region (F(3,72) = 254.91, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed a significant difference between distant sections and contralateral sections at all measuring sites. There was also a significant difference between sections within the infusion area and contralateral sections in the GCL, IML, and MML. Notably, there was also a significant difference between distant sections and sections within the infusion area in the MML and OML (two-way ANOVA, P < 0.05). (Q) Immunostaining of p-ser240/244 ipsilateral to stimulation in a section within the U0126 injection site. (R) High-magnification image of Q. Note the reduction of rpS6 phosphorylation in the dendritic laminae. (S) Immunostaining of p-ser240/244 contralateral to the stimulation. (T) High-magnification image of S. Scale bars: E, 200 µm; F, 50 µm.

Fig 5: The FLCN–mTORC1–TFE3 pathway is separable from the canonical mTOR–S6K pathway. (A, top) Immunofluorescence staining of TFE3 in control (NTC) or FLCN knockout cells. Cells were starved in amino acid-free DMEM for 1 h or starved and restimulated with amino acids or 10% dFBS for 2 h, as indicated. (Bottom) The graph shows quantification of the percentage of cells with nuclear TFE3. (*) P < 0.05 versus NTC - amino acid/- serum. (B) Western blot to phospho-S6K Thr389. Preadipocytes isolated from FLCNlox/lox mice were infected with adenovirus harboring GFP (mock) or Cre to knock out FLCN. Cells were then starved or starved and restimulated with amino acids, as indicated. Total S6K and actin were used as loading controls. (C) Western blot to phospho-rpS6 Ser240/244 in iWAT depots isolated from FLCNlox/lox or FLCN adipKO mice. Animals were fasted overnight and refed ad lib for 4 h. Quantification of phospho-rpS6 is shown at the right, normalized to total rpS6. (*) P < 0.05 by Student's t-test. (D) Immunofluorescence staining of phospho-S6 (red) and TFE3 (green) in control (NTC) cells and cells lacking TSC1 or TSC2. Cells were starved or starved and restimulated with amino acids as indicated. TSC1 or TSC2 genes were deleted by the CRISPR/Cas9 system. (E) Western blot to phospho-S6K. Control and TSC1 or TSC2 knockout preadipocytes were starved or starved and restimulated with amino acids, as indicated (dashed lines indicate separation within the same gel). (F) Diagram of separable mTORC1 hubs. Values are represented as mean ± SEM. Bars, 50 µm.