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. 2020 Jun 25;181(7):1596–1611.e27. doi: 10.1016/j.cell.2020.05.053

Figure 3.

Figure 3

Oncogenic PIK3CA Drives Enhanced Arachidonic Acid Metabolism

(A and B) Arachidonic acid (AA) levels measured by REIMS in MCF10A PIK3CA WT and MUT cells cultured under full 5% horse serum or fatty acid-free (FAF) conditions for 72 h (n = 3 biological replicates) (A). AA levels of 18 breast PDX tumors (n = 9 PIK3CA WT and n = 9 MUT) (left) (B). Three sections corresponding to different tumor regions were analyzed with REIMS. Data are summarized in the boxplot to the right.

(C) 12 primary breast tumors (n = 5 PIK3CA WT and n = 7 MUT) (left). Data are summarized in the boxplot to the right.

(D) Breast, ovarian, pancreatic, sarcoma, and colorectal PDX tumors (n = 5 PIK3CA WT and MUT tumors for breast, pancreatic, sarcoma, and colorectal tissues, and n = 4 PIK3CA WT and MUT ovarian PDX tumors).

(E) Heatmap and Venn diagram summarizing the intracellular eicosanoids that were significantly different between MCF10A PIK3CA WT and MUT cells. Rows correspond to the Z score scaled eicosanoid intensities detected by LC-MS (n = 3 biological replicates).

(F) Heatmap and Venn diagram summarizing the eicosanoids of the conditioned media (CM) that were significantly different between MCF10A PIK3CA WT and MUT cells.

(G) Cell proliferation assays of MCF10A PIK3CA WT cells cultured in CM derived from WT or H1047R MUT cells before or after lipid depletion (LD), with or without the supplementation of 25 μM AA, palmitate, or palmitoleate.

(H) Cell proliferation assays of MCF10A PIK3CA H1047R MUT cells before or after LD, with or without the supplementation of 25 μM AA, palmitate, or palmitoleate. Sulforhodamine B (SRB) protein staining was used in (G) and (H) to measure cell proliferation over 5 days (replicates from n = 3 wells). Error bars in (G) and (H) represent mean ± SEM for each time point. p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. p values in (A)–(D) were calculated with unpaired, two tailed Student’s t test. Two-way ANOVA was used for (G) and (H).