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Fig. 3 | Cellular & Molecular Biology Letters

Fig. 3

From: Senescence-associated reprogramming induced by interleukin-1 impairs response to EGFR neutralization

Fig. 3

SW48-CXR exhibits a lower number of Ki67-positive cells when treated with TRAP IL-1 and higher IL-1R1 and SNAI1 protein expression compared with SW48. Sensitive and resistant SW48 cell lines were treated for 4 days as follows: SW48 with control medium (10% FBS), IL-1 (α and β, 10 ng/ml each), and CTX (50 μg/ml) alone and in combination with TRAP IL-1 (20 μg/ml) (a), while SW48 CXR cells were treated with control medium (10% FBS + CTX 50 μg/ml) and TRAP IL-1 (20 μg/ml) (b). Cells were incubated with anti-Ki67 antibody and with DAPI (nuclei). Number of Ki67-positive nuclei was calculated through ImageJ software from three different fields taken for each sample. Histograms show the percentage of Ki67-positive nuclei of one representative of two independent experiments. Statistical analysis was carried out using two-way ANOVA with Tukey’s multiple comparisons test and t-test in a and b, respectively, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 29 mm. In the upper panel, SW48 cells were seeded in flask and treated with CTX (50 μg/mL) from day 0 to day 4. Immunoblots were processed with the indicated antibodies. In the lower panel, SW48 cells were treated with control medium, IL-1 (α and β 10 ng/ml each), CTX (50 μg/ml), and TRAP IL-1 (20 μg/ml), and SW48 -CXR cells were treated with control medium (FM, with CTX 50 μg/ml) and TRAP IL-1, for 4 days. Protein extracts were incubated with anti IL-1R1 and SNAI1. Signals were quantified and normalized (numbers below each lane). GAPDH was used as loading control protein (c). SW48-CXR cells were maintained under CTX treatment (as described in “Materials and methods”). The next day, medium was changed, and the indicated treatment was applied (TRAP IL-1 20 μg/ml) for 2 days. Thereafter, mRNA was extracted, and gene expression was determined for IL1R1 and SNAI1 (d). Statistical analysis was carried out using two-way ANOVA with Sidak’s multiple comparisons test ***p < 0.001; ****p < 0.001

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