Fig. 2

A Representation of the different SORL1 proximal promoter constructs (P1-7) used in luciferase assays with their corresponding molecular lengths (bp). B P3 is a responsive promoter sequence to Hrg β-1 stimulation. A control empty vector (EV) and P1-7 were individually expressed in BT-474 cells together with pRL-TK Renilla luciferase transfection control and cells were stimulated or not with 20 ng.mL^–1 Hrg β-1 for 24 h. Shown are luciferase activities relative to the control sample (EV, PBS-treated). C Representative confocal microscopy images of P3-GFP promoter reporter construct-expressing BT-474 cells treated or not for 24 h with 20 ng.mL⁻¹ Hrg β-1 in the presence or absence of 100 nM trametinib. Scale bars: 10 µm. D Quantification of the mean intensity of GFP signal per cell (whole-cell area). N = 36 cells per group. Three biological replicates. E Trametinib inhibits Hrg β-1-induced upregulation of SorLA. BT-474 cells were cotreated with 20 ng.mL⁻¹ Hrg β-1 and either the pan-AKT inhibitor MK-2206 (2 µM) or the ERK pathway inhibitor trametinib (100 nM) for 48 h. Representative immunoblotting of SorLA, AKT(p)S473, total AKT, ERK1/2(p)T202/Y204, total ERK1/2, with GAPDH as a loading control. F Quantification of SorLA levels normalized to loading control and relative to non-treated cells. GSORL1 mRNA levels, relative to HPRT1, determined with RT-qPCR in BT-474 cells stimulated or not with 20 ng.mL⁻¹ Hrg β-1 in the presence or absence of trametinib relative to non-treated cells. Data are mean ± SD from four (B) and three (F and G) independent biological replicates; statistical analysis: (B) Two-way ANOVA, Dunnett’s multiple comparisons test. (F and G) Student’s t test (unpaired, two-tailed, unequal variance). (D) Box plots represent median and interquartile range, and whiskers extend to maximum and minimum values; One-way ANOVA, Dunn’s multiple comparisons test.