1. Volume 23, Issue 5, Pages 867-880 (May 2016)
Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells 
Jonathan L. Coloff, J. Patrick Murphy, Craig R. Braun, Isaac S. Harris, Laura M. Shelton, Kenjiro Kami, Steven P. Gygi, Laura M. Selfors, Joan S. Brugge 
Cell Metabolism 
Volume 23, Issue 5, Pages (May 2016)
DOI: /j.cmet
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Cell Metabolism 2016 23, 867-880DOI: (10.1016/j.cmet.2016.03.016)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
MCF-10A 3D Suspension Culture as a Model of Proliferation and Quiescence
(A) Model of MCF-10A morphogenesis in 3D suspension culture.
(B) Representative histogram of DNA content of day-5 and day-15 MCF-10A cells.
(C) Cell accumulation over time in 3D suspension culture. Values are the mean ± SEM of three independent experiments.
(D) Representative images of day-5 and day-15 MCF-10A structures stained for Ki67, GM130, or LAMC2 (red) and DAPI (blue). Scale bar, 50 μm.
(E and F) Consumption and/or secretion of glucose and lactate (E), glutamine, glutamate, and NH4+ (F) in day-5 and day-15 MCF-10A cells.
Values are the means ± SEM of three independent experiments. ∗p < 0.05 (Student’s t test); #p < 0.05 (one-way ANOVA). N.S., not significant.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 2
Metabolomic and Proteomic Analysis of Proliferating and Quiescent Mammary Epithelial Cells
(A) Heatmap of statistically significantly changed metabolites (p < 0.05) between day-5 and day-15 cells.
(B) Heatmap of statistically significantly changed proteins (p < 0.05) between day-5 and day-15 cells.
(C) Heatmap of all identified protein products of a PCNA-associated gene expression signature. Arrows mark canonical proliferation markers Ki67 and PCNA.
(D) Histograms of the day-15/day-5 ratio of all identified metabolites, proteins, or metabolic proteins.
(E) The top ten enriched pathways from integrated pathway analysis of metabolite and protein changes from day 5 and day 15. Dashed line represents p value of 0.05.
(F) Pathway model of all identified metabolites and proteins involved in glutamate-to-αKG conversion. Values are the means ± SD of triplicate samples. ∗p < 0.05 (Student’s t test).
See also Figure S1.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 3
Changes in Transaminases and GLUD in Proliferation and Quiescence
(A) Representative western blot for GLUD and transaminases during MCF-10A morphogenesis in 3D suspension culture.
(B) Transaminase and GLUD mRNA levels during MCF-10A morphogenesis in 3D suspension culture. Values are the means ± SEM from three independent experiments.
(C–E) Activity assays for GLUD (C), alanine transaminase (D), and aspartate transaminase (E) in proliferating and confluent MCF-10A cells grown in 2D culture. Values are the means ± SEM of three independent experiments.
(F) Proliferation index, transaminase, and GLUD1 mRNA expression as measured by microarray in TEBs and ducts from 5-week-old mice. Each data point is from an individual mouse, and error bars indicate the means ± SD from five mice.
∗p < 0.05 (Student’s t test); #p < 0.05 (one-way ANOVA). N.S., not significant.
See also Figure S2.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 4 Analysis of NEAA Synthesis Using 15N Isotope Tracing
(A) Analysis of 15N incorporation into amino acids in proliferating and confluent cells cultured with [α-15N]-glutamine for 24 hr. Values are the means ± SEM of two independent experiments.
(B) Aspartate and alanine labeling normalized to labeled glutamate after culture with [α-15N]-glutamine for 24 hr. Values are the means ± SEM from two independent experiments.
(C) [α-15N]-glutamine incorporation into intracellular glutamine over time. Values are the means ± SD of triplicate samples from an experiment representative of two independent experiments.
(D) Glutamine labeling after culture with [α-15N]-BCAAs for 24 hr. Values are the means ± SEM from two independent experiments.
∗p < 0.05 (Student’s t test). N.S., not significant.
See also Figure S3.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 5
The Relationship between GLUD and Transaminases in Breast Cancer
(A) NH4+ secretion in inducible GLUD1 MCF-10A cells treated with or without dox. Values are the means ± SD of triplicate samples from an experiment representative of two independent experiments.
(B) Analysis of 15N incorporation into aspartate and alanine in MCF-10A cells ± GLUD1 overexpression. Values are the means ± SD of triplicate samples from an experiment representative of two independent experiments.
(C) MCF-10A cell accumulation over time ± GLUD1 overexpression. Values are the means ± SEM from three independent experiments.
(D) Size of structures grown in traditional MCF-10A 3D culture. Values are the means ± SD of >190 structures analyzed per condition from an experiment representative of two independent experiments. The underlying contour plot shows the distribution of structure size.
(E) Tumors with PTEN and/or GLUD1 homozygous deletion from all TCGA cancer studies or breast only (copy number data from 27 TCGA studies of tumors of distinct tissue origin).
(F) Correlation of GLUD1 mRNA and copy number in all CCLE cell lines.
(G) Representative western blot for GLUD1/2 in inducible GLUD1 HCC-1937 cells treated with or without dox for 24 hr.
(H) HCC-1937 cell accumulation over time ± GLUD1 overexpression. Values are the means ± SEM from two independent experiments.
(I) Analysis of 15N incorporation into aspartate and alanine in HCC-1937 cells ± GLUD1 overexpression. Values are the means ± SD of triplicate samples from an experiment representative of two independent experiments.
∗p < 0.05 (Student’s t test); #p < 0.05 (two-way ANOVA).
See also Figure S4.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 6 Transaminase and GLUD Expression in Human Breast Tumors
(A) Correlation of transaminase and GLUD mRNA expression with the 11-gene proliferation signature in TCGA breast tumor samples.
(B) mRNA levels (RSEM × 1,000) of transaminases and GLUD in the four major breast cancer molecular subtypes. Lum, luminal.
(C and D) 11-gene proliferation signature score (C) and PSAT1 promoter methylation (D) in the four major breast cancer molecular subtypes.
(E–G) PSAT1 mRNA levels (RSEM × 1,000) (E), promoter methylation (F), and correlation with proliferation in ER-negative breast tumors (G).
(H) PSAT1 mRNA correlation with proliferation in diverse TCGA datasets.
Statistical significance in (A), (G), and (H) was determined by Pearson correlation (in H, ∗p < 0.05), ANOVA (B–D), or Student’s t test (E and F).
See also Figure S5.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions

9. Figure 7
The PI3K/mTOR Pathway Promotes PSAT1 Expression and Contributes to GLUD Suppression in Proliferating Cells
(A) Transaminase mRNA levels in TCGA breast tumors with normal or high (Z score >2) levels of 4EBP1 S65 phosphorylation (data are log2 transformed).
(B) Western blot of S6 phosphorylation during MCF-10A morphogenesis in 3D suspension culture.
(C) GOT1, GOT2, GPT2, and PSAT1 mRNA levels after treatment of day-4 MCF-10A cells in 3D culture with Torin1 for 24 hr. Values are the means ± SEM from four independent experiments.
(D) Representative western blot after treatment of day-4 MCF-10A cells in 3D culture with Torin1 for 24 hr.
(E) mRNA levels of day-5 and day-15 MCF-10A cells ± dox-inducible expression of myrAkt1. Values are the means ± SEM from two independent experiments.
(F) Representative western blot of day-5 and day-15 MCF-10A cells in 3D culture with or without dox-inducible expression of myrAkt1.
(G) Representative western blot of MCF-10A cells plated sparsely in 2D culture (50,000 per well in a six-well plate), densely in 2D (300,000 per well), or after 4 days of growth in 3D suspension culture treated with or without Torin1 for 24 hr. An arrow marks the validated GLUD band.
(H) GLUD activity assay on MCF-10A cells plated sparsely or densely in 2D culture with or without Torin1 for 24 hr. Values are the means ± SEM from two independent experiments.
(I) NH4+ secretion in proliferating MCF-10A cells treated with BEZ-235 (BEZ) for 24 hr. Values are the means ± SEM of three independent experiments.
∗p < 0.05 (Student’s t test). N.S., not significant.
See also Figures S6 and S7.
Cell Metabolism  , DOI: ( /j.cmet )
Copyright © 2016 Elsevier Inc. Terms and Conditions