Figure 4
Validation of RNA-seq analysis. (a) Immunofluorescence staining of Caco-2 cells showed enhanced L1CAM (green, arrow in v) and integrin beta-1 expression (gray, arrow in vii) in Neo1 transfected cells (v–viii) when compared to control cells (i–iv). Fibronectin (red, arrowhead in ii) was more globular in control cells while fibrils are clearly visible in Neo1 transfected cells (red, arrow in vi) suggesting cell migration. Co-localization (yellow) of L1CAM (green, arrowhead in i) and fibronectin (red, arrowhead in ii) can also be clearly seen in endosome-like structures. Scale bar-20 μm. (b) Western blots and densitometric analysis show increased FN1 expression in Caco-2 cells after Neo1 knockdown. Each band represents cell lysate proteins from a biological replicate from three independent experiments. Full length blot for FN1 and GAPDH are represented in Supplementary Fig. S5. (c) Immunofluorescence staining of Caco-2 cells reveals the vesicular distribution of intracellular MMP1 and also showed enhanced staining in Neo1 treated cells, Scale bar-20 μm. (d) q-PCR validation of RNA-seq analysis. RT-qPCR of top 4 genes under cell migration category after Neo1 knockdown in co-transfected Caco-2 cells shows significant upregulation of CEACAM1, ANXA1 and downregulation of NOX1 relative to the reference gene (TBP). Data represent mean ± SEM of 6 biological replicates. p-value = 0.03 for CEACAM1, p-value < 0.0001 for ANXA1 and NOX1. L1CAM levels were not significantly changed, p-value = 0.67 (two-tailed student’s t-test).
