Fig. 3

Signaling components essential for the AA-stimulated retrograde trafficking. All cells are HeLa cells. a Cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 µM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. b Endogenous SLC38A9 was depleted by lentivirus-transduced shRNAs as assessed by RT-qPCR from n = 3 independent experiments. c The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. Knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for phospho-S6K1 (p-S6K1) and GAPDH. d SLC38A9 is required for the AA-simulated Golgi trafficking. Cells treated with indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a. e Immuno-blots showing that endogenous Lamtor1 and RagA/B were depleted by respective lentivirus-transduced shRNAs. f Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was similar to d. g, h Under Lamtor1 knockdown condition similar to e, an RNAi-resistant Lamtor1 was able to express and rescue the AA-stimulated Golgi trafficking. i Lamtor1 is required for the AA-stimulated reduction of surface CD8a-furin-mEos2. Knockdown and surface labeling were similar to e and Fig. 2j, respectively. Surface intensity was normalized by mEos2 total intensity. Surface DMEM/HBSS-ratio is the normalized surface intensity under DMEM divided by that under HBSS treatment. j mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin, or 250 nM Torin1 was present throughout the treatment. In a, b, d, f, h–j, the displayed value is the mean of n = 3 independent experiments and individual data points are shown as red dots. Error bar, mean ± s.d.; P values were from t test (unpaired and two-tailed); N.S., not significant (P > 0.05); *P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA