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. 2019 Nov 12;219(1):e201901097. doi: 10.1083/jcb.201901097

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Figure 6. — ¹⁵N-leucine incorporation is heterogeneous among lysosomes in single HeLa cells. (A) Images of HeLa cells expressing Lamp1-APEX2 metabolically labeled with ¹⁵N-leucine for 4 d before washout for 54 h and fixation. Arrows indicate lysosomes with high incorporation of ¹⁵N-leucine. Arrowhead indicates a filipodia, which also exhibits high ¹⁵N-leucine incorporation. HSI image represents the ¹²C¹⁵N/¹²C¹⁴N ratio as a hue and ion counts as an intensity for each pixel. Representative images from a single cell are shown. (B) Quantification of ¹⁵N-leucine incorporation into areas of cytosol or individual Lamp1-APEX2–positive lysosomes for single cells treated as described in A. (C) Quantification of ¹⁵N-leucine incorporation into areas of cytosol, nucleus, or individual lysosomes (identified by Lamp1-APEX2 labeling) for experiment described in A. Dotted line indicates natural abundance of ¹²C¹⁵N relative to ¹²C¹⁴N. In B and C, four cells (one HeLa^WT and three HeLa ATG5 KO) were analyzed (in two technical and one biological replicates). (D) Incorporation of ¹⁵N-leucine and ³²S abundance of individual lysosomes in experiment described in A. Four cells (one HeLa^WT and three HeLa^{ATG5 KO}) were analyzed (in two technical and one biological replicates). (E) HeLa cells expressing mito-APEX2 were labeled as in A and ¹⁵N-leucine incorporation into areas of cytosol, nucleus, or individual lysosomes (identified by ³²S intensity) was measured. Dotted line indicates natural abundance of ¹²C¹⁵N relative to ¹²C¹⁴N. A total of three HeLa^WT cells were analyzed (in one technical/biological replicate). Error bars in all graphs represent SD. Scale bar in all images = 1 µm.