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. 2019 Mar 4;129(4):1626–1640. doi: 10.1172/JCI98288

Figure 4. An intact autophagy pathway and lysosomal amino acid sensing and transport are necessary for delayed activation of mTOR by mutant ATP6V1B2.

Figure 4

(A and B) ATG7–/– HEK293T cells were generated using CRISPR-Cas9 targeting, and independent single-cell clones were expanded. Control ATG7 WT cells were expanded from cells that were subjected to CRISPR-Cas9 targeting but remained intact for ATG7. All clones were subsequently infected with lentiviruses carrying doxycycline-inducible WT or mutant ATP6V1B2. Cells were induced with doxycycline for 72 hours, and cell lysates were prepared for immunoblotting using the indicated antibodies. (B) Results of quantification (p-S6K/t-S6K) of 4 independent experiments using data from all lanes labeled WT, Y371C, and R400Q. Statistical analysis compared the difference of the differences between measurements under ATG7 WT and ATG7–/– conditions (see Methods). P = 0.06 for Y371C and P = 0.04 for R400Q for ATG7–/–/ATG7 comparisons (standard regression-based Wald-type tests). Error bars indicate the SD. (C and D) SLC38A9–/– HEK293T cells were generated using CRISPR-Cas9 targeting, and independent single-cell clones were expanded. Control SLC38A9 WT cells were expanded from cells that were subjected to CRISPR-Cas9 targeting but remained intact for SLC38A9. All clones were infected with lentiviruses carrying doxycycline-inducible WT or mutant ATP6V1B2. Cells were induced with doxycycline for 72 hours, and cell lysates were prepared for immunoblotting using the indicated antibodies. (D) Results of quantification (p-S6K/t-S6K) of 3 independent experiments using data from all lanes labeled WT, Y371C, and R400Q. Statistical analysis compared the difference of the differences between measurements under SLC38A9 WT and SLC38A9–/– conditions (see Methods). P = 0.01 for Y371C and P = 0.01 for R400Q for SLC38A9–/– and SLC38A9 comparisons (standard regression-based Wald-type tests). (B and D) *P < 0.05, by standard regression-based Wald-type tests. Error bars indicate the SD.