Fig. 5

Effects of miR-29b, LAMC1 and PPIC perturbation on motility and invasion of LM-MEL-45. a In the absence of a chemotactic gradient, migration of LM-MEL-45 cells into a central detection zone was decreased by treatment with a miR-29b-3p mimic, and partially phenocopied by siRNA-mediated knock-down of either LAMC1 or PPIC (data represent mean + SEM relative to controls of independent triplicates, all treatments at 10nM). b Following reverse transfection with the indicated agents (final concentrations all 10nM), and overnight culture on agarose, LM-MEL-45 spheroids were embedded into a bovine collagen type I matrix and allowed to invade over 24 h prior to staining with a green-fluorescent viable cell dye and imaging (representative spheroids shown). Treatment with a miR-29b-3p mimic virtually ablated any invasion of cells into surrounding collagen, whilst siRNA-mediated knockdown of PPIC led to marked invasion and/or lack of cohesion, seen as a pronounced spheroid halo. c Cross-sectional cellular density measurements of representative spheroids (see also Figure AF5.6 within Additional file 5) confirm relative lack of invasive cells at the spheroid surface in miR-29b-3p mimic treated cells (second panel) and, to a lesser extent, in LAMC1 knockdown cells (third panel). PPIC knockdown cells displayed a broad spheroid cell density profile consistent with extensive cellular egress into surrounding collagen (bottom panel). d Quantitation of the invasion distance from spheroid edge confirmed significant decrease in collagen invasion following miR-29b-3p overexpression, and increase following PPIC knockdown. Error bars show mean ± SEM of at least seven spheroids per treatment