Fig. 4

ALK downstream signalling pathway is enhanced upon ALKAL2 activation and dampened upon CZB treatment in cms1 cells. A ALK downstream signaling activity in SW48 (CMS1) cells treated with conditioned medium containing ALKAL1, ALKAL2 or their combination for either 20′ or 30′. pALK/ALK and pAKT/AKT quantification is provided. ⍺-tubulin was used as loading control. B Investigation of ALK and its downstream pathway activation upon stimulation with ALKAL1/2 containing medium for 30′ in LoVo cells, with or without 30′ CZB pre-treatment. Quantification of pALK and pAKT is shown. C Evaluation of ALK, pERK and pAKT inhibition in starved LoVo (CMS1) cells, following treatment with CZB 1 μM over time, from 1 to 6 h. β-actin along with total AKT and ERK2 were used as loading control. D ALK expression and activation assessment in LoVo (CMS1) and HCT116 (CMS4) cells in basal conditions and upon 30′ and 60′ CZB 1 μM treatment. β-actin was used as loading control. E Droplet-digital PCR performed on RNA extracted from cells belonging to CMS4 (HCT116), CMS3 (HT29) and CMS1 (LoVo, SW48 xenografts). The expression of ALK and ALKAL2 is reported as copy number in 100 ng of RNA, normalized for B2M expression. F Gelatin-degradation assay performed on SW48 treated with ALKAL1/2-containing medium. Cells were stained with Phalloidin-TRITC (F-Actin) and DAPI (nuclei) prior to visualization. Invasion was quantified in terms of area of degraded gelatin (black spots visible in the green channel) normalized over the nuclei count in the same field. At least 7 fields were analyzed for each condition. Number of cells analyzed, CTR: n = 843, ALKAL1: n = 569, ALKAL2: n = 268 and ALKL1 + 2: n = 491. Results are reported as fold increase related to control. One-way ANOVA test was applied. Scale bar: 50 μm