Fig 1: ADCY10 PKA-dependently determines ferroptosis sensitivity in LUAD cells. (A) Endogenous glutamate phosphorylates GFPT1 via PKA. H1975-based GGG cells were under the same treatment as that in Figure 2C, before further treating with or without erastin (10 µM), in the presence or absence of H89 (10 µM) and Rp-cAMPs (200 µM) for 8 h. Indicated proteins were analyzed by IB. (B) GFPT1 activity was analyzed by GDH method in H1975-based GGG cells under the same treatment as that in panel A. (C-F) PKA boosts erastin-induced ferroptosis. A549 and H1975 cells ectopically expressing PKACα were treated with erastin (10 µM), in the presence or absence of PKI (10 µM), Fer-1 (1 µM) or DFO (80 µM). Cell viability (C) and cell death (D) were analyzed 24 h after treatment, and lipid ROS (E) and MDA generations (F) were analyzed 16 h after treatment. (G) ADCY10 level were analyzed in 90 paired LUAD and their adjacent specimens. (H) WT or ADCY10-/- H1975-based GGG cells were under the same treatment as that in Figure 2D but treating with or without erastin (10 µM) for 8 h. Relative GFPT1 activity was analyzed utilizing GDH method, and ADCY10 was analyzed by IB. The level of proteins was normalized to that of GAPDH, and the normalized level of proteins in DMSO-treated cells was arbitrarily set to 1. (I-K) H1975-based GGG cells were pretreated with or without EGTA (0.1 mM), Dox (1 µg/ml) and EGCG (5 µM) for 24 h before further treating with erastin (10 µM) for 8 h. Relative Ca2+ (I) and cAMP concentration (J), and GFPT1 activity (K) were then measured. (L-M) ADCY10 expression correlates with reduced GFPT1 activity and induced cAMP following erastin (10 µM) treatment for 8 h. Reduced GFPT1 activity (L) and induced cAMP (M) were shown as the percentage to the DMSO-treated control, and ADCY10 was measured by IB in indicated LUAD cells. (N) Representative micrograph of LUAD tissue sections with different ADCY10 level treating with erastin (10 µM) with or without Fer-1 (1 µM) for 24 h. ADCY10 level was measured by IHC. Ferroptotic cells and nuclei were visualized using PI (red) and DAPI (blue). PI positive area from each sample was also calculated as the percentage to the whole section and graphed on the right. Scale bar, 1 mm. (O) MDA concentration was measured in the same sample as that in the panel N (n = 20/group). The data are shown as the mean ± SD from three biological replicates (including IB). **P < 0.01 indicates statistical significance. Data in B, C, D, E, F, H, I, J, K, N, O were analyzed using a one-way ANOVA test. Data in G were analyzed using a Student's t test. Data in L, M were analyzed using the Spearman rank-correlation analysis.