Journal: Theranostics

Article Title: Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma

doi: 10.7150/thno.55482

Figure Lengend Snippet: GFPT1 is critical for endogenous glutamate to determine ferroptosis sensitivity. (A) Schematic overview of the HBP signaling from glucose to O-GlcNAcylation. (B) Endogenous glutamate affects HBP metabolites. Indicated metabolites were measured in H1975-based GGG cells under the same treatment as that in Figure D. (C) Relative GFPT1 activity was measured in GGG cells under the same treatment as that in Figure D but treated with or without erastin (10 µM) for 8 h. The GFPT1 activity was normalized between those treated with erastin and DMSO. (D) Metabolites downstream of GFPT1 restore YAP alteration by erastin. p-YAP S127 , O-YAP T241 and YAP were measured by IB in H1975 cells treated with or without erastin (10 µM), glucose (25 mM), GlcN (5 mM) or GlcNAc (5 mM) for 8 h. (E-F) GlcN and GlcNAc diminish ferroptosis sensitivity. LUAD cells were treated with erastin (10 µM) with or without Fer-1 (1 µM) or DFO (80 µM), in the presence or absence of GlcN (5 mM) or GlcNAc (5 mM). Cell death was measured after treatment for 24 h (E). MDA was measured after treatment for 16 h (F). (G) GFPT1 S205A restores YAP alteration by erastin. FLAG-GFPT1 S205A was ectopically expressed in H1975 cells prior to the treatment with or without erastin (10 µM) for 8 h. Indicated proteins were analyzed by IB. (H) GFPT1 boosts protein stability of YAP. CHX (10 µg/ml) chase experiments were performed in H1975 cells with or without GFPT1 overexpression or knockout. The relative protein levels of YAP were shown as the ratios between YAP and GAPDH, and the “0 h” points were arbitrarily set to 100%. (I) GFPT1 reduces ubiquitination of YAP. The ubiquitination of YAP was measured in control cells and H1975 cells with or without GFPT1 overexpression or knockout using anti-Ub antibodies following immunoprecipitation using anti-YAP antibodies. IgG antibodies were used as negative controls. (J-K) Restore of GFPT1 diminishes ferroptosis sensitivity. FLAG-GFPT1 S205A was ectopically expressed in LUAD cells before treatment with erastin (10 µM) with or without Fer-1 (1 µM) or DFO (80 µM). Cell death was measured after treatment for 24 h by SYTOX green staining followed by flow cytometry (J). MDA was measured after treatment for 16 h (K). The data are shown as the mean ± SD from three biological replicates (including IB). **P < 0.01 indicates statistical significance. Data in B were analyzed using a one-way ANOVA test. Data in C, E, F, J, K were analyzed using a two-way ANOVA test.

Article Snippet: The antibodies used for co-IP were: anti-FLAG (CST, #8146 or #2368), anti-YAP (Santa Cruz Biotechnology, #sc-101199), anti-βTrCP (Abcam, #ab233638) and anti-IgG (CST, #3900).

Techniques: Activity Assay, Over Expression, Knock-Out, Immunoprecipitation, Staining, Flow Cytometry