Fig 1: The effect of IL-1ß, TNF-a, Shh treatment in combination with si-KRAS on KRAS down-stream molecules (p-/t-ERK1/2 and p-/t-AKT1) and Ras enzymatic activity in the MT-KRAS cell lines (SW1990 and Panc-1) and WT-KRAS cell line (BxPC-3). The protein expression of p-/t-ERK1/2 and p-/t-AKT1 at Shh treatment in combination with si-KRAS treatment (A); The effect of combined IL-1ß or TNF-a with si-KRAS transfection on p-/t-ERK1/2 and p-/t-AKT1 (B); Ras enzymatic activity at Shh treatment in combination with si-KRAS treatment (C); The effect of combined IL-1ß or TNF-a with si-KRAS transfection on Ras enzymatic activity (D) [a. SW1990, b. Panc-1, c. BxPC-3]. All data were obtained in three independent experiments. *p < 0.05; **p < 0.01.
Fig 2: Results of the pGL3-8×Gli1 BS luciferase reporter assay in the MT-KRAS cells (SW1990 and Panc-1) and WT-KRAS cells (BxPC-3) after treatment with: IL-1ß, TNF-a alone or in combination with si-KRAS transfection (A). Shh alone or in combination with si-KRAS transfection (B). All data were obtained in three independent experiments. *p < 0.05; **p < 0.01.
Fig 3: Neutrophil accumulation involves in intestinal tumor deterioration in KRAS-mutated CRC; A, APC-WT and APC-KRASG12D mice at 8th, 12th, 16th and 20th weeks were euthanized. Tumor number was evaluated by counting single polyps in small colons. B, The number of neutrophils counted using flow cytometry, Single-cell suspensions were prepared from the peripheral blood (a), spleen (b), BM(c) and mLN (d) of APC-WT and APC-KRASG12D mice euthanized at indicated ages. The graphs show total numbers or frequencies of CD45.2 þ Ly6G þ CD11b þ neutrophils in respective organs. C, The content of G-CSF in the serum of APC-WT and APC-KRASG12D mice. * p < 0.05, n = 8. The measurement data was expressed as mean ± standard deviation. Data analysis at different time points was performed by repeated measurement ANOVA, follows by Bonferroni post hoc test
Fig 4: MiRNA-96-5p directly binds to the 3’UTR of HBEGF and KRAS mRNAs in BUMPT cells. (A) TargetScan database analysis shows putative miR-96-5p binding sites in the 3’UTR of HBEGF and KRAS mRNAs. (B, C) Dual luciferase reporter assay results show relative luciferase activities in BUMPT cells co-transfected with luciferase reporter vectors carrying wild-type (WT) or mutant (MUT) 3’ UTR’s of (B) HBEGF and (C) KRAS (C) plus miR-96-5p or miR-NC. (D, E) RT-qPCR analysis shows (D) HBEGF and (E) KRAS mRNA levels in BUMPT cells transfected with 100 nM miR-96-5p mimics or miR-NC, and then treated with TGF-ß1 for 24h. (F) Representative western blot images and (G, H) densitometric measurements show the levels of (G) HBEGF and (H) KRAS proteins in BUMPT cells transfected with 100 nM miR-96-5p mimics or miR-NC, and then treated with TGF-ß1 for 24h. Note: The data are expressed as means ± SD (n = 6). # denotes p < 0.05 when comparing miR-NC plus TGF-ß1or miR-96-5p mimic with saline groups vs. miR-NC plus saline group; * denotes p < 0.05 when comparing miR-96-5p mimics plus TGF-ß1group vs. miR-NC plus TGF-ß1 group and KRAS-WT or HBEGF-WT plus miR-96-5p mimic vs. other groups.
Fig 5: In vivo, ex vivo, and in vitro verification of attenuation of Kras-induced senescence by Arid1a deficiency.(A) Representative images of senescence-associated beta-galactosidase (SA-ß-Gal) staining of frozen pancreatic sections from KC mice and AKC mice. (B) SA-ß-Gal-positive lesions were counted at five random fields under the microscope in four KC mice and one AKC mouse, presented as percentages. (C) Representative images of SA-ß-Gal staining of ex vivo culture from KC and AKC mice 1 month after administration of tamoxifen. (D) Quantification of the intensity of SA-ß-Gal staining at five random fields under the microscope. (E) Colony formation assay of ARID1A knockout cells and wildtype human pancreatic Nestin-expressing (HPNE) cells with KRAS induction by doxycycline (6 µg/ml) for 15 days. (F) Quantification of colony number in panel (E). The colony formation assay was performed twice. Student’s t-test: **p<0.01; ***p<0.001. Scale bars: 200 µm.
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