Fig 2: SLC36A1 increases SLC38A9 expression and promotes the translocation of SLC38A9 to LAMP2-positive lysosomal clusters. (A) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct to overexpress SLC36A1. The mRNA level of SLC38A9 was analyzed by qRT-PCR. (B) C2C12 cells were transfected with siRNA to knockdown SLC36A1. The mRNA level of SLC38A9 was analyzed by qRT-PCR. (C) Immunoblotting analysis of protein samples from (A,B) with anti-SLC38A9 or anti-β-Actin antibody. (D) Densitometric analysis of the immunodetection of SLC38A9 relative to β-Actin loading control. (E) C2C12 cells were transfected with pcDNA3.1-SLC36A1 construct or siRNA to overexpress or knockdown SLC36A1 and stained with anti-SLC38A9 or anti-LAMP2. Scale bar: 10µM. (F) Ratio of SLC38A9 to LAMP2 (%). To assess SLC38A9 translocation, the numbers of SLC38A9 and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Fig 3: IL28A expression was activated by ATG10S and ATG10 mutants with Cys44-Cys135 mutation, and mediated autolysosome formation and HCV degradation. (A) Protein levels of the immune factors (IL28A, IRF3, and IRF7) and autophagy flux-related proteins (LC3B, P62, and LAMP2) were analyzed by Western blotting. (B) Transcription levels of IL28A, IRF3, and IRF7 were tested by qPCR. (C) Co-Immunoprecipitation showed the interactions among LAMP2, IL28A, P62, and LC3B using anti-LAMP2 antibody, and the interactions among IL28A with ATG10 mutant proteins and LAMP2 using anti-Flag antibody (labeling the ATG10, ATG10S, and the ATG10 mutants). (D) Immunofluorescence analysis shows co-localization of IL28A with LAMP2 by using anti-IL28A and anti-LAMP2 antibodies and co-localization of ATG10 mutants with IL28A by using anti-Flag and anti-IL28A antibodies in the HCV subreplicon cells. (E) The interactions between autophagosomes and lysosomes were disappeared in IL28A-knockdown cells by immunoprecipitation with anti-LC3B antibody (upper two panels). The replication of HCV subgenomic replicon was restored in IL28A-knockdown cells detected by Western blotting and RT-PCR tests in which HCV CORE and NS5B proteins and core RNA were obviously elevated via IL28A downregulation (lower two panels). Scale bars, 15 μm. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the replicon model.

Fig 4: Effects of Arg and Leu on the location of mTOR on LAMP2-positive lysosomal clusters, activation of mTORC1 and expression of SLC38A9 and SLC36A1 in C2C12 cells. (A) C2C12 cells were pretreated without serum for 15 h and without amino acids for 3 h then cultured for 10 min in a special medium (Supplemental Table S1) including the no amino acids group (−AA), the only Arg group (+Arg), and the only Leu group (+Leu). Cells were stained with anti-mTOR (A2445, Abclonal Technology, Wuhan, China) or anti-LAMP2. Scale bar: 10 µM. (B) Ratio of mTOR to LAMP2 (%). To assess mTOR translocation, the numbers of mTOR and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. (C) Immunoblotting analysis of protein samples from (A) with anti-mTOR, anti-phospho-mTOR, anti-S6K, anti-phospho-S6K, anti-SLC38A9, anti-SLC36A1, or anti-β-Actin antibody. (D) Densitometric analysis of the immunodetection of mTOR relative to β-Actin loading control. (E) Densitometric analysis of the immunodetection of phospho-mTOR relative to β-Actin loading control. (F) Densitometric analysis of the immunodetection of p70S6K relative to β-Actin loading control. (G) Densitometric analysis of the immunodetection of phospho-p70S6K relative to β-Actin loading control. (H) Densitometric analysis of the immunodetection of SLC38A9 relative to β-Actin loading control. (I) Densitometric analysis of the immunodetection of SLC36A1 relative to β-Actin loading control. (J) The mRNA levels of SLC38A9 and SLC36A1 were analyzed by qRT-PCR. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.

Fig 5: SLC38A9 increases SLC36A1 expression and promotes the translocation of SLC36A1 to LAMP2-positive lysosomal clusters. (A) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct to overexpress SLC38A9. The mRNA level of SLC36A1 was analyzed by qRT-PCR. (B) C2C12 cells were transfected with siRNA to knockdown SLC38A9. The mRNA level of SLC36A1 was analyzed by qRT-PCR. (C) Immunoblotting analysis of protein samples from (A,B) with anti-SLC36A1 or anti-β-Actin antibody. (D) Densitometric analysis of the immunodetection of SLC36A1 relative to β-Actin loading control. (E) C2C12 cells were transfected with pcDNA3.1-SLC38A9 construct or siRNA to overexpress or knockdown SLC38A9 and stained with anti-SLC36A1 or anti-LAMP2. Scale bar: 10µM. (F) Ratio of SLC36A1 to LAMP2 (%). To assess SLC36A1 translocation, the numbers of SLC36A1 and LAMP2-positive spots per cell were calculated using IPP6.0 and Image J software. Values are the mean ± SEM; n = 3; * p < 0.05; ** p < 0.01.