Figure 1
From: Dysregulation of Blimp1 transcriptional repressor unleashes p130Cas/ErbB2 breast cancer invasion
High expression of Blimp1 protein in p130Cas/ErbB2 invasive acini is sustained by Erk1/2 activation. (A) Day 14 culture of control (Mock) and p130Cas overexpressing MCF10A.B2 (Cas) plated on a matrigel:collagen matrix and treated with either 1µM ethanol (vehicle) or AP1510 on day 10 to activate ErbB2 (ErbB2; Cas/ErbB2). Scale bars, 50µm. Magnification 20X. White arrowheads indicate multiacinar structures. Black arrowheads indicate invasive acini. (B) On day 14 acinar structures were recovered and lysed and Blimp1 mRNA expression levels were evaluated by RT-PCR. Statistical analysis was performed using the Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Day 14 protein extracts were probed for Blimp1 in a western blot analysis. Tubulin was used as the loading control. (D) Densitometric analysis of protein levels of at least three independent experiments is shown (mean ± s.e.m). Blimp1 protein modulation was calculated relative to Mock level and normalized on tubulin as the loading control. Statistical analyses were performed using the Student’s t-test (**p < 0.01, ***p < 0.001). (E) Acini derived from Mock and Cas cells were treated on day 10 with either DMSO (vehicle), 10 µM LY294002 (PI3K inhibitor) or 25 µM PD98059 (MAPK pathway inhibitor) in both in the presence and absence of 1 µM of AP1510. After 4 days of treatment, acini were recovered and lysed. Total cell extracts were probed for Blimp1 in a western blot analysis. GAPDH was used as loading control. (F) Densitometric analysis of protein levels of at least three independent experiments is shown (mean ± s.e.m). Protein modulation was calculated relative to Mock level and normalized to GAPDH as the loading control. Statistical analyses were performed using the Student’s t-test (*p < 0.05, **p < 0.01). (G) Representative phase images of day 14-acinar structures showing invasive protrusion impairment upon AKT/PI3K and MAPK pathway inhibition. Scale bars, 50 µm. Magnification 20X. White arrowheads indicate multiacinar structures. Black arrowheads indicate invasive acini.
