Fig. 6: Knockout of IL-36R expression in CT26 cells decreases tumour burden in vivo. | Oncogene

Fig. 6: Knockout of IL-36R expression in CT26 cells decreases tumour burden in vivo.

From: IL-36 signalling enhances a pro-tumorigenic phenotype in colon cancer cells with cancer cell growth restricted by administration of the IL-36R antagonist

Fig. 6

A IL-36R Knockout CT26 cells were generated by CRISPR-Cas9 technology. This was confirmed by (i) PCR detection of exon 5 of IL-36R, (ii) western blotting for IL-36R protein, (iii) sanger sequencing of exon 5-spanning primer products with TIDE analysis and (iv) functional analysis of cellular response to IL-36 agonist stimulation by CCL2 gene expression in scramble control cells and IL-36R knockout cells. B Scramble control or IL-36R KO CT26 cells (2 × 105) were subcutaneously inoculated into BALB/c mice (n = 6 per group) and tumour growth measured as previously described. Data points represent the mean value ± SEM with final tumour volumes also shown. C Tumour tissue was extracted, processed and analysed for immune cell infiltration by flow cytometric analysis for CD4 T cells (CD4+/CD8−), CD8 (CD4−, CD8+), Neutrophils (CD45+, LY6G+), and Macrophage (CD45+, F4/80+) quantification. D Tumour tissue was extracted, processed, paraffin embedded and immunostained with anti-Ki67 and images were taken of invasive margins and tumour cores and quantified by ImageJ software. Representative images are shown (mag = 40X; Scale 100 µm). Statistical analysis was performed by One-way ANOVA with Dunnetts post hoc test (A) and student T Test (B, C, D).

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