To further validate that CD43 promotes cell transformation leading to tumor development, and considering that the signaling capacity of CD43 depends on its intracellular domain

Unbiased of the mobile line, reduction of CD43 expression AFQ-056 racemate stages did not avert tumor formation, considering that all mice (n=8) inoculated with CD43low cells developed tumors (Figure S4C). However, impairing CD43 expression resulted in significantly smaller tumors, independently of the tissue origin (lung, < 50% cervix, < 80%) as compared to tumors derived from cells expressing normal CD43 levels (Figure 2A and 2B, lower panel). However, reducing CD43 expression in the colon DLD-1 tumor cells only partially affected tumor size (<30%) (Figure 2C, lower panel). Altogether, these results show that CD43 expression Figure 1. CD43 signaling cooperates with oncogenic signals to promote cell transformation. NIH-3T3-hEGFR (A) or E6/E7 transgenic mouse fibroblasts (C) carrying the pFNeo empty vector (pFNeo), expressing wild-type CD43 (Wt) or CD43 lacking the intracellular domain (IC) were grown to confluence the monolayer was then wounded (t=0) and healing was evaluated by light microscopy at the indicated time points. NIH-3T3-hEGFR fibroblasts were grown in soft agar as described in material and methods after three weeks, colonies were counted (B). E6 transgenic mouse fibroblasts stably transfected with the indicated constructs were grown to confluence for three weeks foci were stained with Giemsa and counted (D). 3X106 E6/E7 fibroblasts stably transfected were injected subcutaneously into nu/nu mice one month later, animals were sacrificed and tumor mass was weighed (E). Data shown are representative of at least four independent experiments performed with at least four independent pFNeo, Wt or IC clones from each cell line. Graphs represent the average cell number SD of three independent experiments using at least 3 independent clones. p < 0.05 vs pFNeo.Figure 2. CD43 expression confers tumoral fitness to human tumor-derived cells. A549 lung (A), CasKi cervix (B) or DLD-1 colon (C) tumor cells containing the empty pSuper (pSup) vector or expressing the CD43 specific RNAi (RNAi) were cultured to confluence, the monolayer was then wounded and healing was evaluated (upper panel). Cells were also cultured in soft agar as indicated in material and methods, after three weeks colonies were counted (middle panel). Cells (1X106 for A549, 3X106 for CasKi or DLD-1) were injected subcutaneously into nu/nu mice one month later, animals were sacrificed and tumor weight was evaluated (lower panel). Data represents the average SD of four independent experiments performed with four independent pSup or RNAi clones for each cell line. p < 0.05, p < 0.01 vs pSup potentiated the tumorigenic capacity of non-hematopoietic human tumor derived cell lines.To further validate that CD43 promotes cell transformation leading to tumor development, and considering that the signaling capacity of CD43 depends 3625714on its intracellular domain [19], we transfected a mutant form of CD43 lacking the intracellular domain in A549 cells expressing endogenous CD43 and tested whether this mutant would work as a dominant negative molecule, reducing wound healing and anchorage-independent cell growth capacities.