Fig. 1
Generation and characterization of TRAP-overexpressing and TRAP knockdown MDA-MB-231 breast cancer cells. TRAP protein expression and enzymatic activity in MDA-MB-231 breast cancer cells overexpressing TRAP (TRAP1low, TRAP2low, TRAP3high, TRAP4high) compared to the mock-transfected control (ctrl) (a-c) and in TRAP knockdown cells (sh2 and sh3 + 4) compared to the scrambled control (scr) (d-g). TRAP knockdown was established by sh-hairpin technology in the TRAP3high clone and initially tested by TRAP mRNA quantification (d, n = 3). One representative Western blot of TRAP staining of lysate and medium (monomeric TRAP 5a: 37 kDa, cleaved TRAP 5b: 16 kDa and 25 kDa) together with the normalization control β-Actin (42 kDa, only in lysate) is shown (a, e). Measurements in media are normalized to cell number and time in culture. Differential TRAP protein expression of the replicate means of biological replicates was quantified by densitometry (b, n = 3; f, n = 3). TRAP enzymatic activity, is normalized to total protein expression for lysates or number of cells and time in culture for medium (c, n = 7; g, n = 6). Statistical comparison was performed on biological replicates by Kruskal-Wallis test (Fig. 1b ) or ANOVA test (Fig. 1c , d , f , g ). Groups are generally compared to their respective controls (ctrl or scr) and significance is annotated with an asterisk (*). Specifically, the comparison of TRAP protein in medium between the high and low expressing cells TRAPlow and TRAP4high is annotated with a hash (#). “n=” indicates the number of replicates