Fig 2: AF upregulated p21 to trigger cell cycle arrest, senescence, and apoptosis in rat cardiomyocytes. (a) Western blotting determined p21, CDC25, and Cyclin B1 protein levels in myocardial tissues; (b) immunohistochemistry assay determined SA-β-gal and γ-H2AX levels in myocardial tissues; (c) TUNEL staining evaluated cell apoptosis. N = 6. An independent sample t-test was used for comparisons between two groups. **p < 0.01.

Fig 3: SOX6 silencing downregulated p21 and mitigated cell cycle arrest, senescence, and apoptosis in rat cardiomyocytes. Rats were injected with si-SOX6 plasmids via tail veins on day 14 before AF modeling. (a) RT-qPCR measured SOX6 mRNA level in rat myocardial tissues; (b) Western blotting measured SOX6, p21, CDC25, and Cyclin B1 protein levels in rat myocardial tissues; (c) immunohistochemistry assay determined SA-β-gal and γ-H2AX protein levels; (d) TUNEL staining assessed cell apoptosis. N = 6. An independent sample t-test was conducted for comparisons between two groups. **p < 0.01.

Fig 4: Functional characterization and MEK inhibitor sensitivity of RASGRF2 fusions.a Structure of five OCLN–RASGRF2 fusions, as in Fig. 2d. Three distinct fusion variants (v1–v3) were identified. b Western immunoblotting of lysates from HEK 293 T cells expressing the indicated cDNAs to assess cellular levels of active RAS (GTP-RAS) and ERK activation (p-ERK). OCLN–RASGRF1 is included as a positive control and comparator for RASGRF2 fusion constructs. OCLN–RASGRF2 v1 contains the epitope recognized by the RASGRF2 antibody but not the occludin antibody. OCLN–RASGRF2 v3 contains the epitope recognized by the occludin antibody but not the RASGRF2 antibody. Molecular weight is indicated in kDa on the left. c Anchorage-independent proliferation of NIH3T3 cells expressing the indicated cDNAs in soft agar assays. Cells were cultured in soft agar for 19 days. Images are representative of three independent experiments. The white marker denotes 300 μm. d Quantification of average colony area for NIH3T3 cells expressing the indicated cDNAs (normalized to GFP) after 19–21 days. Mean and standard error for three independent experiments (with three replicates each) are shown. These data were collected in parallel with those shown in Fig. 4d using the same GFP control. *, P < 0.05. **; P < 0.01 by two-tailed t-test compared to GFP. NS not significant (compared to GFP). e Western immunoblotting of lysates from NIH3T3 cells expressing the indicated cDNAs. f Proliferation of Ba/F3 cells expressing the indicated cDNAs after withdrawal of IL3 at Day 0. The experiment was performed 3 times. g Western immunoblotting of lysates from Ba/F3 cells expressing the indicated cDNAs. Cells expressing GFP were cultured in the presence of 1 ng/ml IL3. Cells expressing the other cDNAs were cultured in the absence of IL3. h Ba/F3 cells expressing the indicated cDNAs were treated with trametinib at the indicated concentrations. After 4 days, cell viability was determined with Cell Titer-Glo. Cells expressing GFP were cultured with 1 ng/mL IL3. Mean and standard error are shown, and the experiment was performed 3 times. i Western immunoblotting of lysates from Ba/F3 cells expressing OCLN–RASGRF2 v1 or OCLN–RASGRF2 v3 treated with trametinib at the indicated concentrations for 24 h after serum starvation for 4 h. Cl PARP cleaved PARP.

Fig 5: Genetic features of RASGRF fusions that contribute to RAS activation and cellular transformation.a Structure of full-length RASGRF1 and N-terminal truncation constructs lacking the PH1 domain (R1 del1–139), PH1 and DH domains (R1 del1–422), and constructs preserving only the REM and CDC25 domains (R1 del1–622) or only the CDC25 domain (R1 del1–784). b Western immunoblotting of lysates from HEK 293 T cells expressing the indicated cDNAs to assess cellular levels of active RAS (GTP-RAS) and ERK activation (p-ERK). Molecular weight is indicated in kDa on the left. c Western immunoblotting of lysates from HEK 293 T cells transiently transfected with the indicated cDNAs. LINGO1–R1, LINGO1–RASGRF1. d Tumor cell focus formation in NIH3T3 cells expressing the indicated cDNAs. Cells were cultured for 5 weeks. Images are representative of foci identified in 3 independent experiments. Black marker denotes 300 μm. e Quantification of NIH3T3 cell foci transduced with the indicated cDNAs after 4–6 weeks. Mean and standard error for three independent experiments (with three replicates each) are shown. ***; P < 0.0001 by two-tailed t-test compared to GFP. f Western immunoblotting of lysates from NIH3T3 cells expressing the indicated cDNAs. g Western immunoblotting of lysates from HEK 293 T cells transiently transfected with the indicated cDNAs.