1 Supporting Information Sequestration-mediated downregulation of ...
Supporting Information
Sequestration-mediated downregulation of de novo purine biosynthesis by AMPK Danielle L. Schmitt1, Yun-ju Cheng2, Junyong Park2, and Songon An1* 1
Department of Chemistry and Biochemistry, 2 Department of Mathematics and Statistics,
University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250, USA
1
Supporting Information Figure S1. Immunostaining of endogenous FGAMS after AICAR treatment. (A) Distribution of endogenous FGAMS in non-transfected HeLa cells. (B) Distribution of endogenous FGAMS (red) with FGAMS-EGFP (green) in transfected HeLa cells. Scale bar, 10 µm.
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Supporting Information Figure S2. No effect of AICAR on localization of free-standing EGFP. Transiently expressed EGFP alone in HeLa cells remained diffuse throughout the cell after 5 hr incubation with 300 μM AICAR (A and B). Scale bar, 10 µm.
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Supporting Information Figure S3. Immunocytochemistry showing AMPKα in HeLa cells. Total AMPK (A, C and E) and phospho-Thr172 AMPK (B, D and F) are immunostained using monoclonal antibodies without drug treatment (A and B) and after 4 hr incubation with 200 µM AICAR (C and D) or 170 µM metformin (E and F). A differential interference contrast image of (B) is presented as an inset to indicate the boundary of the cell dotted in (B). Scale bar, 10 µm.
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Supporting Information Figure S4. No effect of metformin on localization of EGFP-ATIC. EGFP-ATIC remained diffusive in the cytoplasm after treatment with 170 μM metformin for 4 hr (A and B). Scale bar, 10 µm.
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Supporting Information Figure S5. Cluster size analysis of fluorescent images. Distribution of cluster sizes of the FGAMS-EGFP self-assemblies (A, N = 84) and the purinosomes (B, N = 72) indicates both clusters are similar in size, with an average cluster size of 0.46 μm2 for FGAMSEGFP self-assembly, and 0.52 μm2 for purinosomes.
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Supporting Information Figure S6. AICAR-induced FGAMS-EGFP clusters in the presence of other cellular structures. The self-assemblies of FGAMS-EGFP were induced with 200 μM AICAR in HeLa cells. Live cells expressing FGAMS-EGFP (A, D and G) were stained for lysosomes (B), mitochondria (E) and lipids (H) using LysoTracker Red, MitoTracker Red and Dil Stain, respectively. In addition, fixed cells expressing FGAMS-EGFP (J) were stained for autophagosomes using an antibody of endogenous LC3 (K). Merged images (C, F, I and L) indicated that the AICAR-induced FGAMS self-assemblies are different from lysosomes, mitochondria, lipid-bound organelles and autophagosomes. Scale bar, 10 µm.
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Sequestration-mediated downregulation of de novo purine biosynthesis by AMPK Danielle L. Schmitt1, Yun-ju Cheng2, Junyong Park2, and Songon An1* 1
Department of Chemistry and Biochemistry, 2 Department of Mathematics and Statistics,
University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, MD 21250, USA
1
Supporting Information Figure S1. Immunostaining of endogenous FGAMS after AICAR treatment. (A) Distribution of endogenous FGAMS in non-transfected HeLa cells. (B) Distribution of endogenous FGAMS (red) with FGAMS-EGFP (green) in transfected HeLa cells. Scale bar, 10 µm.
2
Supporting Information Figure S2. No effect of AICAR on localization of free-standing EGFP. Transiently expressed EGFP alone in HeLa cells remained diffuse throughout the cell after 5 hr incubation with 300 μM AICAR (A and B). Scale bar, 10 µm.
3
Supporting Information Figure S3. Immunocytochemistry showing AMPKα in HeLa cells. Total AMPK (A, C and E) and phospho-Thr172 AMPK (B, D and F) are immunostained using monoclonal antibodies without drug treatment (A and B) and after 4 hr incubation with 200 µM AICAR (C and D) or 170 µM metformin (E and F). A differential interference contrast image of (B) is presented as an inset to indicate the boundary of the cell dotted in (B). Scale bar, 10 µm.
4
Supporting Information Figure S4. No effect of metformin on localization of EGFP-ATIC. EGFP-ATIC remained diffusive in the cytoplasm after treatment with 170 μM metformin for 4 hr (A and B). Scale bar, 10 µm.
5
Supporting Information Figure S5. Cluster size analysis of fluorescent images. Distribution of cluster sizes of the FGAMS-EGFP self-assemblies (A, N = 84) and the purinosomes (B, N = 72) indicates both clusters are similar in size, with an average cluster size of 0.46 μm2 for FGAMSEGFP self-assembly, and 0.52 μm2 for purinosomes.
6
Supporting Information Figure S6. AICAR-induced FGAMS-EGFP clusters in the presence of other cellular structures. The self-assemblies of FGAMS-EGFP were induced with 200 μM AICAR in HeLa cells. Live cells expressing FGAMS-EGFP (A, D and G) were stained for lysosomes (B), mitochondria (E) and lipids (H) using LysoTracker Red, MitoTracker Red and Dil Stain, respectively. In addition, fixed cells expressing FGAMS-EGFP (J) were stained for autophagosomes using an antibody of endogenous LC3 (K). Merged images (C, F, I and L) indicated that the AICAR-induced FGAMS self-assemblies are different from lysosomes, mitochondria, lipid-bound organelles and autophagosomes. Scale bar, 10 µm.
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