Skip to main content
. Author manuscript; available in PMC: 2018 Feb 26.
Published in final edited form as: Cell Rep. 2018 Feb 6;22(6):1365–1373. doi: 10.1016/j.celrep.2018.01.030

Figure 4. LSTase Activity of CPT1A Promotes Hematopoietic Cell Proliferation Under Glutamine Depletion.

Figure 4

(A) Lysates from 293T cells stably expressing vector control, CPT1A WT, and CPT1A G710E were western blotted with anti-LVxx(succ)K, anti-CPT1A, and anti-β-actin antibodies.

(B) CPTase activity of membrane proteins from 293T cells stably expressing vector control, CPT1A WT and G710E.

(C) FAO activity in 293T cells expressing vector control, CPT1A WT, and G710E.

(D) WB of lysates from Ba/F3 cells stably expressing vector control, CPT1A WT, H473A, and G710E with anti-LVxx(succ)K, anti-CPT1A, anti-enolase 1, and anti-β-actin antibodies.

(E) Enolase activity of lysates from Ba/F3 cells stably expressing vector control, CPT1A WT, H473A, and G710E.

(F) GC/MS analysis of succinyl-CoA extracted from Ba/F3 cells expressing vector control, CPT1A WT, H473A, and G710E.

(G) FAO activity in Ba/F3 cells expressing vector control, CPT1A WT, H473A, and G710E.

(H) Glutamine-independent proliferation of Ba/F3 cells stably expressing vector control, CPT1A WT, H473A, and G710E.

(I) Enolase activity of lysates from Ba/F3 cells treated with vehicle control and 0.5 μM ENOblock.

(J) Glutamine-independent proliferation of Ba/F3 cells treated with vehicle control and 0.5 μM ENOblock.

(K) WB of lysates from BT474 cells stably expressing vector control and CPT1A shRNA with anti-succinylated lysine, anti-CPT1A, anti-enolase 1, and anti-β-actin antibodies.

(L) GC/MS analysis of succinyl-CoA extracted from BT474 cells stably expressing vector control and CPT1A shRNA.

(M) GC/MS analysis of enolase activity by measuring the conversion rate of [3-13C]-2PG to [3-13C]-PEP in cells incubated with [U-13C6]-glucose.

(N) Proliferation of BT474 cells expressing vector control and CPT1A shRNA in complete medium.

(O) Proliferation of BT474 cells expressing vector control and CPT1A shRNA in glutamine-free medium.

Error bars, ±SD of three independent measurements. P values were determined by a two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. See also Figure S4 and Table S4.