Fig 2: The silencing of KIFC1, LRRC59, and TRIP13 causes a mesenchymal-epithelial morphological transition in MDA-MB-231 cells that is accompanied by a strong impairment of cell motility. MDA-MB-231 cells were treated with control siRNA (siCTRL) or siRNA targeting KIFC1, LRRC59, and TRIP13 (siKIFC1, siLRRC59, and siTRIP13). After 72 h, the cells were evaluated for their morphology (optical microscope) (A) and motility using wound healing assays (B). The scale bar represents 50 µm. The bars indicate the mean ± S.D. (n = 3, each point is a technical duplicate). Statistical significance was assessed with Student's t test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). The evaluation of silencing efficacy was performed by lysing the cells in SDS-sample buffer after the wound healing assay and analyzing the expression of the three proteins by Western blot. Representative WB analyses are shown together with red ponceau stained membranes to verify total protein normalization. Relative protein expression levels were obtained by densitometric analyses of WB analyses (siCTRL versus siKIFC1, siLRRC59, and siTRIP13). The bars indicate the mean ± S.D. (n = 3). Statistical significance was assessed with Student's t test (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

Fig 3: The expression of KIFC1, LRRC59, and TRIP13 is linked to HMGA1. MDA-MB-231 cells were treated with control (siCTRL) or HMGA1-targeting siRNAs (siA1_1 and siA1_3) for 72 h. A, mRNA and protein expression levels of the indicated genes were analyzed by qRT-PCR and Western blot. Gene expression levels in HMGA1-silenced cells were compared with that of siCTRL cells. GAPDH was used for normalization. Representative WB analyses are shown together with red ponceau stained membranes to verify total protein normalization. The histogram graphs relative to Western blot analyses were obtained using densitometric analyses (siCTRL versus siA1_1 and siA1_3). The bars indicate the mean ± S.D. (n = 3). Statistical significance was assessed with Student's t test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). B, Immunohistochemistry analyses performed on breast cancer specimens (IDC, invasive ductal carcinoma; DCIS, ductal carcinoma in situ; IDC/DCIS, mixed IDC and DCIS) positive for KIFC1, LRRC59, and TRIP13 and HMGA1.