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Figure 6: The association of EMT-related transcription factors with CD44 and CD44v6. (a) Flow cytometry analysis of EMT-related transcription factors in A818.4 wt, -v6kd, and Tspan8kd cells; mean ± SD of 3 experiments; significant differences between wt and CD44v6kd cells: , significant differences between wt and Tspan8 cells: s; (b) WB of EMT transcription factors in wt and CD44v6kd cells; a representative example of 3 repetitions is shown; (c) qRT-PCR of CD44v6, NOTCH, and Nanog in A818.4-wt and -CD44v6kd cells with dependence on stimulation by PMA or CD44v6-crosslinking or CD44ICD transfection or in the presence of TAPI and GSI to block CD44ICD liberation; RQ values (mean ± SD of 3 triplicates, 2 repetitions); significant differences to unstimulated wt cell mRNA: , nt: not tested; (d) WB of EMT transcription factors coimmunoprecipitating with CD44v6 in wt cells and with CD44 in CD44v6kd cells; (E) WB of EMT-related transcription factors in the cytosolic and the nuclear fractions of wt and CD44v6kd cells, which were stimulated by PMA or CD44v6- (wt cells)/CD44 (CD44v6kd cells)-crosslinking; (e and d) representative examples of 3 repetitions; (f) Flow cytometry of EMT-related transcription factor expression in wt, CD44v6kd, and Tspan8kd TEX; mean ± SD (3 assays) of stained LB; significant differences between wt and CD44v6kd TEX: , significant differences between wt and Tspan8 TEX: s. Several EMT-related transcription factors are associated with CD44v6 in cells and TEX, where particularly NOTCH expression becomes upregulated by CD44v6 stimulation. CD44v6 stimulation also supports nuclear transfer of associated EMT transcription factors.