Fig 2: Restoration of CALD1 reverses the effects of miR-1278 on GC cell growth and the MAPK signaling cascade. (a) Western blotting analysis of the CALD1 protein expression in CALD1-overexpressed AGS and GTL-16 cells transfected with an miR-1278 mimic. (b) Cell viability in CALD1-overexpressed AGS and miR-1278 mimic-transfected GTL-16 cells was analyzed using a CCK-8 kit. (c and d) Cell migration and invasion in CALD1-overexpressed AGS and miR-1278 mimic-transfected GTL-16 cells were analyzed using a Transwell assay. (e) The levels of migration-related proteins (E-cadherin, N-cadherin, and Snail) in CALD1-overexpressed AGS and miR-1278 mimic-transfected GTL-16 cells were analyzed using western blotting. (f) The levels of the MAPK signaling pathway-related proteins (Ras, P38, p-P38, ERK1/2, and p-ERK1/2) in CALD1-overexpressed AGS and miR-1278 mimic-transfected GTL-16 cells were analyzed using western blotting (compared with the empty vector group, **P < 0.001; compared with the mimic-NC group, ++ P < 0.001; compared with the CALD1-OE + mimic group, ^^ P < 0.001).

Fig 3: BRAF inhibitor-induced transcriptional changes upstream of CD274. (A) Cartoon showing the design of CD274-promoter-luciferase constructs used for determining the changes in transcriptional activity postdrug treatment. (B) The box plot representing the luciferase activity determined in the cells post 48 hours of transfection, where 2 Kb promoter sequence correlated with maximum activity. (C) Luciferase activity measured in the cells post 48 hours of BRAF or KRAS knockdown. (D) The bar graph represents the difference in the promoter activity between the T85 and T32 cell lines. (E, F) Luciferase (Promoter) activity correlated with increased BRAF inhibitor concentration in the T68 and T85 cells. (****p<0.0001, ANOVA test). ANOVA, analysis of variance.

Fig 4: Spatial analysis of KRAS-high and KRAS-low niches in PDAC.(A) UMAP visualization of major cell populations identified by Xenium spatial transcriptomics in a human PDAC sample.(B) UMAP visualization of EPCAM, KRAS, AREG, and CD3E expression in the human PDAC tissue of the Xenium spatial transcriptomic data.(C) Spatial distribution of major cell populations in representative tumor regions enriched for KRAS-high versus KRAS-low cancer cells. Cell types are color-coded as indicated. Number of regions: KRAS-high, n=6; KRAS-low, n=6. Scale bar: 50 μm.(D) Box plots displaying the proportional distribution of cell populations in spatial regions of KRAS-high and KRAS-low cancer cells. Number of regions: KRAS-high, n=6; KRAS-low, n=6. Wilcoxon test.

Fig 5: CASTOR1 ablation enhances KRASG12D-driven cellular transformation.(A) Detection of KRAS and KRASG12D proteins by immunoblotting in passage 2 MEFs isolated from KRASWT;C1WT, KRASWT;C1KO, KRASG12D;C1WT and KRASG12D;C1KO mice. The expression of KRASG12D in MEFs was induced by Adeno-Cre infection and KRASG12D protein was examined at day 3 post-infection.(B-C) CASTOR1 ablation enhances KRASG12D-induced foci formation in culture. Mock- or Adeno-Cre-infected cells were seeded at 5×105 cells/well in 10 cm-dish at day 3 post-infection and cultured with daily change of medium. Foci formation was examined at day 14 post-seeding with representative images shown (B) and quantified (C).(D-E) CASTOR1 ablation enhances KRASG12D-induced colony formation in semi-soft agar. Mock- or Adeno-Cre-infected cells were seeded in soft agar at day 3 post-infection with daily change of medium. Colonies were examined at day 14 post-seeding with representative images shown (D) and colonies from Adeno-Cre-infected cells were quantified (E). Statistical analysis was performed by Student’s t-test.