SNX27 depletion prolonged animal survival in a xenograft model. (A and A′) For comparative analysis of SNX27 expression, indicated cell lines were lysed and subjected to immunoblotting using anti-SNX27 and anti-actin antibodies (loading control). (B) Different clones of MDA-MB-231 SNX27KO were obtained after cells were transfected with Cas9 protein complexed with either control sgRNA or sgRNA-targeted SNX27. The knockout efficiency was determined by immunoblotting for SNX27 and vinculin (loading control). (C) Control sgRNA transfected MDA-MB-231 cells and SNX27KO clones were seeded on gelatin-coated coverslips, and after fixation immunostained with the anti-cortactin antibody. DAPI and phalloidin were used to label the nucleus and actin, respectively. Images were acquired with a confocal microscope, and the number of cells forming invadopodia were plotted. The inset is representing the actin-cortactin–rich invadopodia. (N = 3, n = 80; scale bars = 10 µm, inset = 4 µm). The graph represents means ± SEM. Two-tailed Student’s t test. (C′) Control MDA-MB-231 cells and two clones of SNX27KO MDA-MB-231 were plated on Matrigel-coated inserts. The cellular invasion was measured and plotted as described earlier (N = 3). The graph represents means ± SEM. One-way ANOVA, *** P < 0.001, **** P < 0.0001. (D and E) An equal number of control MDA-MB-231 cells and SNX27KO cells were injected in the mammary fat pad (D) or intravenous (E) of the mice. (D) 7 wk after primary tumor removal, animals were sacrificed upon developing secondary tumors, as determined by the general health condition of the animals. The number of days for which animals survived was plotted (n = 5 mice/group). Arrows indicate the presence of secondary metastatic tumors in the lungs and liver. (E) Intravenous injection of control MDA-MB-231 cells and SNX27KO cells was evaluated in terms of the appearance of lung metastasis and survival. Tables in D and E use a scoring system based on the level of tumor infiltration: +, at least one tumor nodule present; ++, multiple (three or more) nodules present. Two-tailed Student’s t test was used to determine significance. LN, lymph node; Mets, metastasis; N, number of experimental repeats.
Figure 7.

SNX27 depletion prolonged animal survival in a xenograft model. (A and A ′ ) For comparative analysis of SNX27 expression, indicated cell lines were lysed and subjected to immunoblotting using anti-SNX27 and anti-actin antibodies (loading control). (B) Different clones of MDA-MB-231 SNX27KO were obtained after cells were transfected with Cas9 protein complexed with either control sgRNA or sgRNA-targeted SNX27. The knockout efficiency was determined by immunoblotting for SNX27 and vinculin (loading control). (C) Control sgRNA transfected MDA-MB-231 cells and SNX27KO clones were seeded on gelatin-coated coverslips, and after fixation immunostained with the anti-cortactin antibody. DAPI and phalloidin were used to label the nucleus and actin, respectively. Images were acquired with a confocal microscope, and the number of cells forming invadopodia were plotted. The inset is representing the actin-cortactin–rich invadopodia. (N = 3, n = 80; scale bars = 10 µm, inset = 4 µm). The graph represents means ± SEM. Two-tailed Student’s t test. (C′) Control MDA-MB-231 cells and two clones of SNX27KO MDA-MB-231 were plated on Matrigel-coated inserts. The cellular invasion was measured and plotted as described earlier (N = 3). The graph represents means ± SEM. One-way ANOVA, *** P < 0.001, **** P < 0.0001. (D and E) An equal number of control MDA-MB-231 cells and SNX27KO cells were injected in the mammary fat pad (D) or intravenous (E) of the mice. (D) 7 wk after primary tumor removal, animals were sacrificed upon developing secondary tumors, as determined by the general health condition of the animals. The number of days for which animals survived was plotted (n = 5 mice/group). Arrows indicate the presence of secondary metastatic tumors in the lungs and liver. (E) Intravenous injection of control MDA-MB-231 cells and SNX27KO cells was evaluated in terms of the appearance of lung metastasis and survival. Tables in D and E use a scoring system based on the level of tumor infiltration: +, at least one tumor nodule present; ++, multiple (three or more) nodules present. Two-tailed Student’s t test was used to determine significance. LN, lymph node; Mets, metastasis; N, number of experimental repeats.

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