Fig 2: 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-ß-Actin antibody. (D) Densitometric analysis of the immunodetection of SLC36A1 relative to ß-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.

Fig 3: 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-ß-Actin antibody. (D) Densitometric analysis of the immunodetection of mTOR relative to ß-Actin loading control. (E) Densitometric analysis of the immunodetection of phospho-mTOR relative to ß-Actin loading control. (F) Densitometric analysis of the immunodetection of p70S6K relative to ß-Actin loading control. (G) Densitometric analysis of the immunodetection of phospho-p70S6K relative to ß-Actin loading control. (H) Densitometric analysis of the immunodetection of SLC38A9 relative to ß-Actin loading control. (I) Densitometric analysis of the immunodetection of SLC36A1 relative to ß-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 4: SLC38A9 and SLC36A1 interact. (A) SLC38A9 was associated with SLC36A1 at the endogenous level as detected by Co-IP: anti-SLC36A1 (sc-161150, Santa Cruz) was used for pull-down, and anti-SLC38A9 (ab81687, Abcam) and anti-SLC36A1 (sc-368553, Santa Cruz) were used for detection. (B) Interaction of SLC38A9 and SLC36A1: C2C12 cells were transfected with SLC38A9 or SLC36A1 in the pCMV-HA vector, and the lysates were prepared and subjected to HA immunoprecipitation followed by immunoblotting for the indicated proteins. (C) 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), the only Leu group (+Leu). The interaction of SLC38A9 and SLC36A1 was detected by Co-IP as (A). (D) Densitometric analysis of the immunodetection of SLC38A9 relative to input control or SLC36A1. Values are the mean ± SEM; n = 3; * p < 0.05.

Fig 5: 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-ß-Actin antibody. (D) Densitometric analysis of the immunodetection of SLC38A9 relative to ß-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.