Fig. 2.

miRNA‐483‐3p promotes the EMT program and stem‐like traits in m‐colospheres. (A) Representative western blot analysis of EMT markers and core TFs in parental CRC264 and CRC327, and in miRNA‐483‐3p‐transduced m‐colospheres (CRC264miR and CRC327miR). Actin β was used as loading control. Densitometric analysis is shown in Fig. S2C (n ≥ 3). (B) Representative immunofluorescent stainings of E‐cadherin (E‐Cad), vimentin (VIM) and ZEB1. Nuclei were counterstained with DAPI. Scale bar, 50 μm (n ≥ 3). (C) In vitro limiting dilution sphere‐forming assay. For each m‐colosphere, plots generated by the elda software are shown, reporting the estimated stem cell frequency (percentage of clonogenic cells) with confidence intervals (C.I.). (D) Representative western blot analysis of stem cell TFs. Densitometric analysis is shown in Fig. S2D (n ≥ 3). (E) Representative western blot analysis of colorectal differentiation markers mucin 1 (MUC1), keratin 20 (CK20) and CDX2. Tubulin β was used as loading control (n = 3). Densitometric analysis is shown in Fig. S2D (n ≥ 3). (F) Differentiation assay. M‐colospheres were cultured for 4 days in pro‐differentiating conditions (adhesive substrate and basal medium containing 10% FBS). Immunofluorescent stainings for CDX2 and CK20 are shown. Nuclei were counterstained with DAPI. Scale bar: 50 μm (N = 3). Bright field acquisition is shown in Fig. S2E.