FIGURE 2 from MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases

PTK6 inhibition increases ubiquitination and degradation of SNAIL in TNBC. A, PTK6 inhibitor (P21d) treatment or PTK6 shRNA downregulates SNAIL in TNBC cells. Human MDA-MB-231 or mouse MMTV-myc cells were treated with P21d for 24 hours and SNAIL expression was assessed by Western blot analysis. Lysates of MDA-MB-231 cells expressing vector control or either of two independent PTK6 shRNA vectors were assessed for SNAIL expression. B, MDA-MB-231 cells overexpressing HA-ubiquitin and Flag-SNAIL were treated with P21d for 8 hours with MG132 cotreatment for the last 4 hours. Lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-HA or FLAG antibody. The result is representative of three independent experiments. C, Representative images of Duolink PLA using MDA-MB-231 cells coexpressing HA-ubiquitin and Flag-SNAIL. Cells were treated with P21d (5 µmol/L) for 24 hours with MG132 cotreatment for the last 4 hours and incubated with antibodies against HA (Ubiquitin) and FLAG (SNAIL) for PLA (red) and Phalloidin for F-actin and counterstaining. PLA signals (red)/cell were quantitated using ImageJ (left). Scale bar, 15 µm. D, Representative images of PLA using MDA-MB-231 cells overexpressing HA-ubiquitin only. Cells were treated with P21d for 24 hours and with MG132 cotreatment for the last 4 hours and incubated with antibodies against HA (Ubiquitin) and SNAIL for PLA (red) and Phalloidin for F-actin counterstaining. PLA signals/cell were quantitated using ImageJ (left). Scale bar, 15 µm. E, Representative images of PLA using MDA-MB-231 cells overexpressing HA-ubiquitin only that were transfected with control or PTK6 siRNA for 48 hours. Cells were treated with MG132 for the last 4 hours and incubated with antibodies against HA (Ubiquitin) and SNAIL for PLA (red) and Phalloidin for F-actin counterstaining. PLA signals/cell were quantitated using Image (left). Scale bar, 15 µm. All the PLA signals were counted by ImageJ with a fixed setting. Data represent mean ± SEM. *, P = 0.05; ***, P = 0.0001 by Student t test.