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- Effect of 2D and 3D growth conditions on expression of signalling GTPase Rac1b in colorectal cellsPublication . Pereira, Joana; Matos, Paulo; Jordan, PeterRac1 is a member of the Rho-family of small GTPases, which stimulates signalling pathways involved in the control of actin filament dynamics and transcriptional activation. We previously identified that Rac1b, an alternative splicing variant containing an extra exon, is overexpressed in a subset of B-Raf-mutated colorectal tumours. Although the Rac1b protein is expressed at low levels in cells, it is mostly in its active GTP-bound state and stimulates the transcription factor NFkB without affecting other classical Rac1 signalling pathways (such as lamellipodium formation or activation of the kinases PAK or JNK). Experimental depletion of Rac1b revealed an essential role in cell cycle progression and survival of colorectal cancer cells cultured under 2D conditions. Because different cell culture conditions can affect signalling pathway organization, we determined mRNA and protein expression levels and subcellular localization of Rac1b in Caco-2 cells grown as either monolayers on plastic dishes or as polarized epithelia on filters or as 3D spheroids in Matrigel. Most significantly, co-culture experiments with the generated polarized epithelia revealed that the presence of fibroblasts promoted loss of epithelial organization and a transient increase in Rac1b protein levels, implicating stromal cells in the regulation of Rac1b production and intracellular localization.