Fig 2: The BLT2 cascade lies downstream of mutant KRAS in lung cancer cells.a BLT2 mRNA expression and 5-/12-LOX protein expression in BEAS-2B normal lung cells and in A549 and SK-LU-1 KRAS-mutant lung cancer cells. The results are representative of three independent experiments with similar results. b Levels of BLT2 cascade proteins in cell lysates of the indicated cell lines. KrasG12D overexpression was induced in BEAS-2B cells by transfection with a KrasG12D expression plasmid. KRAS was knocked down in A549 and SK-LU-1 cells using siRNA-KRAS. Scr scrambled siRNA control. The western blot results are representative of three independent experiments with similar results. Band intensities were quantified using ImageJ and are expressed as the fold change relative to the control value. c A549 cells were treated with an anti-IL-6 neutralizing antibody (100 ng/ml). Cells were then counted with a hemocytometer. The data are presented as the mean ± SD values of three independent experiments. d ELISAs of IL-6 production in cell culture supernatants. A549 cells (left) were treated with the BLT2-specific inhibitor LY255283 (10 μM). BEAS-2B cells (right) were transfected with the KrasG12D expression plasmid. Twenty-four hours after transfection, the cells were exposed to LY255283 (10 μM). The data are presented as the mean ± SD values of three independent experiments. e A549 (left) and SK-LU-1 cells (right) were treated with inhibitors of BLT2, 5-LOX, and 12-LOX (10 μM LY255283, 5 μM MK886, and 800 nM baicalein, respectively) and then counted. The data are presented as the mean ± SD values of three independent experiments. f BEAS-2B cells were transfected with the KrasG12D expression plasmid. Twenty-four hours after transfection, the cells were exposed to the indicated inhibitor and then counted. Cell proliferation data were statistically analyzed at 48 h and compared between the indicated inhibitor groups and the DMSO control group. The data are presented as the mean ± SD values of three independent experiments. The data were analyzed using an unpaired two-tailed Student’s t-test. *p < 0.05.

Fig 3: High expression levels of BLT2 in lung adenocarcinoma patients with KRAS mutations.a–c IHC analysis of KrasG12D and BLT2 expression in lung adenocarcinoma patients (a normal lung tissue; b, c lung adenocarcinoma tissue). Scale bars, 400 μm. d IF analysis of KrasG12D and BLT2 expression in lung adenocarcinoma patients (upper panel, normal lung tissue; lower panel, lung adenocarcinoma tissue). Scale bars, 100 μm.

Fig 4: Accumulation of spontaneous tumorigenic Kras mutations in Sirt2-deficient mice. (a) Accumulation of tumorigenic Kras mutations during inflammation-coupled neoplasm. Mouse pancreas genomic DNA from wild-type and Sirt2 KO, with and without caerulein-treatment, were analyzed for KrasG12D or KrasG12V mutations by competitive allele-specific TaqMan PCR. The table shows the summary of analysis for percentage of mice positive for mutations (46 wild-type mice, 56 Sirt2 KO mice) after pancreatitis. (b) Detail description of wild-type and Sirt2 KO mice with KrasG12D and KrasG12V mutations. (c) Bar graph representation of (b). (d) Immunostaining detection of KRAS-G12D mutant protein in mouse pancreas. Mice pancreas tissues were fixed, embedded in paraffin, and immuno-histochemical staining with anti- KRAS-G12D antibody was performed. Representative images are shown. Bars indicate 100 µm.

Fig 5: Single-cell RNA-Seq analyses of perivascular, glial, and immune cells in KRASG12D-induced mice.(A) t-SNE plot of perivascular cells of AAV-CAG-DIO-MCS– (control) and AAV-CAG-DIO-KRASG12D–treated (KRAS-treated) Cdh5-CreERT2;lsl-tdTomato mice. (B) Representative gene markers, the number of up- and downregulated DEGs, cell numbers (middle), and ratio (right) of each perivascular cell type in control and KRAS-group. (C) Violin plots of marker gene expressions of proliferative fibroblast-like cells (FB). (D) t-SNE plot of microglia/macrophage in control and KRAS group. (E) Representative gene markers, the number of up- and downregulated DEGs, cell numbers (middle), and ratio (right) of each microglia/macrophage cluster in control and KRAS group. (F) Violin plots of representative DEGs in microglia/macrophage clusters in control and KRAS group. (G) t-SNE plot of astrocytes in control and KRAS group. (H) Representative gene markers, the number of up- and downregulated DEGs, cell numbers (middle), and ratio (right) of each cluster of astrocytes in control and KRAS group. (I) Violin plots of representative DEGs in astrocyte clusters in control and KRAS group. (J) t-SNE plot of leukocytes in control and KRAS group. (K) Cell numbers (left) and ratio (right) of leukocytes in control and KRAS group.