Fig. 4.

Unscheduled activation of PERK induces luminal space filling. (A) Whole-cell lysates from 2- to 12-day-old MCF10A acini were immunoblotted with the indicated Abs. The upper right panel shows PERK and Beclin1/ATG6 mRNA transcript levels normalized to GAPDH mRNA from 3D Matrigel acini collected at the indicated time points. Confocal equatorial images from GFP-LC3 (the distribution of LC3-positive events is quantified in Fig. 5A) MCF10A acini fixed at day 8 show punctate basal (arrowheads) and luminal (arrow) LC3 staining (green). Blue, DAPI. Scale bars are 25 μm. (B) Confocal images of day 12 MCF10A Fv2E-ΔNPERK acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP, showing stacked sections to reveal increased cellularity and compaction (upper panels) or equatorial confocal sections of acini (lower panels). The graph (upper right panel) shows the distribution and mean size of day 12 MCF10A Fv2E-ΔNPERK acini treated from day 6 (+) or not treated (−) with 100 pM AP. Size was calculated using SPOT software following the equation [(length × width²)/2 = acinus volume (mm³)] (n = 50). The total number of cells per acinus (lower right panel) was also calculated (n = 50). (C) Fv2E-ΔNPERK acini treated from day 6 to days 9 and 10 (+) or not treated (−) with 100 pM AP were stained with 1 μg/ml EtBr (day 10) or cleaved caspase-3 (Cl-c3) at day 9. Magnifications show intraluminal Cl-c3 staining in nontreated acini versus negative Cl-c3 staining in AP-treated acini. The percentage of apoptotic cells was scored (right graphs) (n = 40). (D) Confocal equatorial images from Fv2E-ΔNPERK MCF10A acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP, showing intraluminal BimEL staining (red). The graph shows the distribution and mean number of intraluminal BimEL-positive cells per acinus. (E) Confocal equatorial images from Fv2E-ΔNPERK acini treated from day 6 to day 12 (+) or not treated (−) with 100 pM AP and transfected with ATF4 or control (scRNA) siRNA. The right graph show the distribution and mean number of intraluminal cells for each equatorial section of a single acinus (n = 20). Scale bar = 10 μm. The right blot shows controls for ATF4 knockdown in the 3D cultures. (F) Equatorial confocal section images of day 20 MCF10A Fv2E-ΔNPERK acini treated from day 6 to day 20 (+) or not treated (−) with 100 pM AP, showing intraluminal filling (left panels). Graphs (right panels) show the mean number and distribution of cells in the outer ring per section (upper panel) and the total number of intraluminal cells per section (lower panel) (n = 50). In all of the images blue shows DAPI staining of nuclear DNA, and unless otherwise indicated, scale bars indicate 25 μm. P values were determined by the t test.