Fig 1: Ineffective XBP1 splicing in LUAD cells facilitates glutamate suppression of GFPT1. (A) IB of GFPT1 in LUAD cells treated with DMSO or erastin (10 µM) for 8 h. The level of GFPT1 was normalized to that of GAPDH, and the normalized level of GFPT1 in DMSO-treated A549 cells was arbitrarily set to 1. (B) Reduced activity and expression of GFPT1 by erastin are closely correlated, and were calculated as the percentage to the ones treated with DMSO for 8 h. (C) GFPT1 expression and activity were analyzed using IB and GDH assay method in WT and GFPT1-/- A549 and H1975 cells. (D) Spliced (s) or unspliced (u) form of XBP1 mRNA in LUAD specimens, as measured by semi-RT-qPCR via agarose gel electrophoresis. (E) Percentage of LUAD specimens with indicated XBP1 splicing conditions. (F) Ineffective XBP1 splicing facilitates GFPT1 decrease. H1975-based GGG cells with or without ectopically expressed XBP1s were under the same treatment as that in Figure 2D but treating with or without erastin (10 µM) for 8 h. MDA-MB-231 cells were treated as the paralleled control. Relative GFPT1 activity was analyzed utilizing GDH assay method. Total and phosphorylated GFPT1 in nuclear and cytosolic fractions were analyzed by IB using normal gels or gels containing phostag™. (G) XBP1 was knocked down in MDA-MB-231 cells before treating with erastin (10 µM) for 8 h. H1975 cells were treated as the paralleled control. GFPT1 activity, total and phosphorylated GFPT1 were analyzed the same way as that in Figure 4F. (H) GFPT1 is phosphorylated at S205 by erastin and Sorafenib. Phosphorylation of indicated GFPT1-FLAG was analyzed by IB using gels containing phostag™ in H1975 cells after treating with or without erastin (10 µM), Sorafenib (5 µM) and RSL3 (5 µM) for 8 h. (I) Phosphorylation at S205 is critical for GFPT1 degradation. The degradation of indicated GFPT1-FLAG was measured by IB in H1975-based GGG cells after pretreating with or without Dox and EGCG for 24 h, before further treating with erastin (10 µM) for indicated hours. GFPT1 level was normalized to that of GAPDH. The data are shown as the mean ± SD from three biological replicates (including IB and semi-RT-qPCR). **P < 0.01 indicates statistical significance. Data in C, F, G were analyzed using a one-way ANOVA test. Data in I were analyzed using a two-way ANOVA test. Data in B were analyzed using the Spearman rank-correlation analysis.
Fig 2: 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 PKACa 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.
Fig 3: qPCR (A) and WB (B, C) were used to detect the effects of AHNAK and NFATC1 knockdown with siRNAs on key glycolysis enzymes, amino acid metabolism enzymes, EMT and PD-L1 immune checkpoints of two bladder cancer cell lines T24 and UMUC3. Glycolysis enzymes involve PFKFB3 and LDHA. Glutamine metabolic enzymes involve GLS and GLUD1. EMT involves E-cadherin and vimentin. ß-actin and a-actin were used as internal references. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, no significance.
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