Fig 2: Exendin-4 stimulates autophagy independently of MTOR and AMPK signaling. (A-C) INS-1E were treated with GLT -/+ Ex4 for 2 h. Western blotting was used to quantify p-MTOR (S2448) (A), p-RPS6 (S240/244) (B) and p-PRKAA/AMPKa (T174) (C) and quantified relative to total MTOR, RPS6 or PRKAA. n = 5 (A and C) or 6 (B). (D) INS-1E were pre-incubated with 10 µM STO-609 or 10 µM Compound C for 1 h prior to the addition of 100 nM Ex4 for 1 h. p-PRKAA (T174) was analyzed by western blotting. Images are representative of three individual experiments. (E and F) INS-1E were pre-incubated with 10 µM STO-609 (E) or 10 µM Compound C (F) for 1 h prior to the addition of GLT -/+ Ex4 for 6 h. LC3 was analyzed by western blotting and quantified as LC3-II:GAPDH ratio. n = 3 (E) or 4 (F). Results are expressed as raw densitometry units corrected for the loading control as stated. All data are mean ± SEM of three to six individual experiments. Statistical analysis was performed using either a one-way (A, B, C) or two-way ANOVA (E and F) followed by Bonferroni’s post-hoc test. *P < 0.05, **P < 0.01 effect of GLT; #P < 0.05, ###P < 0.005 effect of Ex4; ^P < 0.05, ^^^P < 0.005 effect of inhibitor.

Fig 3: rpS6 phosphorylation deficiency increases proliferation and decreases translational fidelity in fibroblasts.(A) Immortalized WT, rpS6P-/-, Akttg and rpS6P-/-;Akttg MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) MEFs described in (A) were seeded in 96-well plates at a density of 4 x103 per well. The proliferation of MEFs described in (A) was monitored as described in (Fig 4B). td, population doubling time. (C) WT and rpS6P-/- immortalized MEFs were incubated for 1 h with 1 mM puromycin, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Quantification of signals obtained in 6 experiments similar to that described in (C). (E) WT, rpS6P-/-, Akttg, and rpS6P-/-;Akttg MEFs were cotransfected with Renilla luciferase expression vector and any one of the following firefly luciferase expression vectors: Fluc(WT), Fluc(Stop) and Fluc(R218S). The transfection efficiency of any of the Fluc constructs was normalized to that of the Renilla luciferase. The relative Fluc activity of each mutant was normalized to Fluc (WT). The result obtained for each mutant in WT MEFs (n = 24) was arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6P-/- MEFs (n = 24) was normalized to that value. Similarly, results obtained for each mutant in Akttg MEFs (n = 12) were arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6P-/-;Akttg MEFs (n = 12) was normalized to that value.

Fig 4: 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 5: Autophagy activation after LPD is similar in muscles and in leukocytes of wild-type mice and col6a1 null mice. (A, B) Western blotting of MAP1LC3B/LC3B in protein lysates of tibialis anterior muscles and blood leukocytes (Leuko) from wild-type and col6a1 null (col6a1-/-) mice fed either with standard diet (SD) or low-protein diet (LPD) for 30 d. Each lane corresponds to one animal. For each condition, animals were injected with 0.4 mg/kg/d of colchicine at the end of the diet, to visualize the autophagic flux. RPS6 was used as a loading control. (C, D) Scatter plots showing linear association (dotted line) between LC3B-II relative intensity in tibialis anterior (TA) and leukocytes from western blot analysis as shown in (A and B). Relative protein levels of LC3B-II in muscles and leukocytes were quantified vs RPS6. Each point corresponds to one animal. Pearson coefficients (R) and P values are shown.