Fig 2: Tumor microenvironment and transcriptome data of KRAS-mutant NSCLC patients with or without KRAS-G12D point mutation. (A-C) IHC analysis of PD-L1 level (A), CD8+ TILs (B), and tumor microenvironment based on PD-L1 and CD8+ TILs (C) according to KRAS oncogene substitutions in a cohort of 112 NSCLC specimens with KRAS mutations. (D) Volcano plot of significantly down- and up-regulated genes in LUAD samples with KRAS-G12D mutation. (E) The distributions of remarkably dysregulated immune-related genes among the five classes based on immune response. (F-G) GO analysis (F) and KEGG analysis (G) of the biological pathways enriched for significantly dysregulated immune-related genes. (H) Heatmap plot of significantly dysregulated immune-related genes. (I) IHC analysis of protein levels of CXCL10 and CXCL11 in LUAD samples with or without KRAS-G12D mutation. Abbreviations: G12D, samples with KRAS-G12D mutation; non-G12D, samples without KRAS-G12D mutation; TIL, tumor infiltrating lymphocyte; GO, Gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; IHC, immunohistochemistry.

Fig 3: Physical interaction between UBIAD1 and H-Ras.a Ectopically expressed UBIAD1 colocalized with H-Ras in the Golgi apparatus. HEK293T cells were transfected with UBIAD1-GFP, Golgi-RFP and BFP-H-Ras, followed by 24 h of cell culture and confocal analysis. The same experiment was repeated three times. b Fluorescence resonance energy transfer (FRET) between UBIAD1 and H-Ras. HEK293T cells were transfected with plasmids as indicated, followed by 24 h of culture, confocal microscopy and PixFRET analysis by ImageJ software. The arrows indicate the FRET signal. Positive control: pCasper-BG (a standard probe showing the BFP-GFP FRET phenomenon). Negative control: BFP and GFP. ***p < 0.001, Student’s t-test, (n = 3 experiments, each with 50 cells). c Interaction of UBIAD1 with H-Ras by co-IP. HEK293T cells were transiently transfected with plasmids as indicated. Forty-eight hours after transfection, the cell lysate was exposed to antibodies and analyzed by IP and IB as indicated. The same experiment was repeated three times. d Interaction of endogenous UBIAD1 with pan-Ras by co-IP. The total cell lysate from HEK293T cells was exposed to rabbit anti-UBIAD1 antibody and analyzed by IP, followed by IB. Rabbit IgG was used as a negative control. The same experiment was repeated three times. e Interaction of endogenous HEIX with pan-Ras in wild-type Drosophila larvae by co-IP. The total cell lysate from larvae was exposed to rabbit anti-HEIX antibody or rabbit IgG and analyzed by IP followed by IB as indicated. The same experiment was repeated three times

Fig 4: CRAF is required for particle macropinocytosis by HRAS transformed cells. (a) Western blotting documenting the downregulation of CRAF by siRNA in RAS3 cells. (b) Flow cytometry illustrates the reduced EV uptake by RAS3 cells following CRAF down-regulation. (c to e) Confocal images documenting the decreased 70-kDa dextran and EV uptake in CRAF siRNA-treated RAS3 cells (c). However, transferrin uptake was not affected in CRAF siRNA treated RAS3 cells (d). Quantification of EV uptake in confocal images; fluorescent deposit counting was conducted in 3 randomly taken images of individual cells

Fig 5: Ras isoforms differentially modulate reciprocal CD40 signaling. a Immunoblot analysis for checking the specificity of siRNA used for silencing respective isoforms. Only the Isoform-specific siRNA but not the control siRNA silenced the Ras isoforms. b Control siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 µg/ml) for 15 min; using Western blot CD40 (3 µg/ml)-induced p38MAPK or ERK-1/2 activation was assessed. c-e siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 µg/ml) for 15 min. siRNAs specific for H-Ras (c), K-Ras (d) and N-Ras (e) modulated the CD40 (3 µg/ml)-induced p38MAPK or ERK-1/2 activation, as assessed by Western blot. e-g siRNAs for H-Ras (f), K-Ras (g) and N-Ras (h) modulated the anti-CD40 antibody (3 µg/ml)-induced IL-12 or IL-10 productions as measured by using specific cytokine ELISA kits, in a macrophage-like mouse cell line P388D1. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 µg/ml) for 48 h. Data represented as mean ± SEM; *, p = 0.05; ** p = 0.005 compared with anti-CD40 stimulated P388D1 cells.