Fig. 3

ErbB3 drives MEC survival through the PI3K-to-Akt signaling pathway during pregnancy. a Mammary glands harvested from 12–15-week-old mice at 16.5 d.p.c., L1, and L5, and stained using IHC for P-Akt Serine 473. Representative images are shown. Original magnification is 400×. Lower panel: percentage of MECs that were P⁺. Each data point represents the average of two random 400× fields from a single tumor; midlines are the average of samples within the group ± SD. P values calculated using Student’s unpaired two-way t test. N = 5. b, c HC11 cells harvested for analysis 72 h after transfection with three distinct siRNA sequences targeting ErbB3, or a scrambled siRNA sequence (siScr). b Western blot analysis using antibodies listed at the left. Representative images are shown, repeated three times. c Caspase 3/7 activity measured using Caspase-Glo, corrected for total protein content, and shown relative to the caspase 3/7 activity measured in cells transfected with siScr sequences. Experiments were assessed in triplicate and repeated four times. Data points are the average of the experimental triplicates; midlines are the average (± SD) of the biological replicates. Student’s unpaired t test. d, e HC11 cells stably transduced with lentivirus encoding ErbB3 shRNA (shErbB3) or shScr, then transduced with Ad.Akt^Myr or Ad.GFP, and harvested for analysis 48 h later. d Western blot analysis using antibodies listed at the left. Representative images are shown, repeated three times. e Cell cultures stained with Annexin V-FITC to detect apoptotic cells. Experiments were assessed in triplicate and repeated four times. Data points are the average of the experimental triplicates across experiments; midlines are the average (± SD) of the biological replicates. Student’s unpaired t test. f, g HC11 cells transduced with Ad.Akt^Myr or Ad.GFP. At 48 h post transduction, cells were treated with BKM120 (1 μM) for 6 h (f) or 16 h (g). f Western blot analysis using antibodies listed at the left. Representative images are shown, repeated three times. g Cell cultures stained with Annexin V-FITC to detect apoptotic cells as described in (e). h, i HC11 cells treated with BKM120 (1 μM) or AZD6244 (1 μM) for 6 h (h) or 16 h (i). h Western blot analysis using antibodies listed at the left. Representative images are shown, repeated three times. i Cell cultures stained with Annexin V-FITC to detect apoptotic cells as described in (e). dpc days post coitus, Lact d lactation day