Figure EV3. TGFβ promotes proline biosynthesis and glutamine oxidation.

• A
  Schematic of stable isotope tracing using l‐glutamine labeled with [¹³C] at all five carbons ([U‐¹³C] glutamine, shown in red).
• B–D
  Kinetic labeling curves of the indicated isotopomers after tracing with [U‐¹³C] glutamine ([U‐¹³C] Gln). NIH‐3T3 cells were treated with TGFβ or mock for 48 h, and the medium was replaced (including all treatments) with DMEM lacking l‐glutamine and supplemented with [U‐¹³C] Gln for the last 2, 8, or 24 h. Metabolites were measured by LC‐MS. For other isotopomers, see Fig 2C–H.
• E
  Western blot of NIH‐3T3 cells treated with TGFβ or mock for the indicated time in the presence of 0.5% FBS. KGA (kidney type) and GAC (glutaminase C) denote the two isoforms of GLS1.
• F
  IMR90 cells were treated with TGFβ or mock for 48 h, and abundance of proline was measured by GC‐MS. Values are relative to mock‐treated cells.
• G
  Tracing of [U‐¹³C] glucose ([U‐¹³C] Glc) into proline. NIH‐3T3 cells were treated with TGFβ or mock for 48 h in DMEM lacking l‐serine and glycine in the presence of 0.5% dialyzed FBS, and the medium was replaced (including all treatments) with DMEM lacking d‐glucose, l‐serine, and glycine and supplemented with [U‐¹³C] Glc for the last 8 or 24 h in the presence of 0.5% dialyzed FBS. Proline was measured by LC‐MS.
• H
  Representative MS1 spectra of the CO1A1 peptide in ECM generated with unlabeled ([¹²C] Gln, left) and fully labeled glutamine ([U‐¹³C] Gln, right). Also see Fig 2K–M.

Data information: P‐values were calculated by two‐sided unpaired t‐test with Welch's correction (F). Bars in (F, G) represent the mean + SD; data in (B–D) represent the mean ± SD. n = 3 (B–D, G); n = 4 (F). A representative experiment is shown (E, H).