Fig. 1.

PERK activation in ECM-detached cells is associated with autophagy induction. (A) Whole-cell lysates from MCF10A cells adhered (A) or suspended for 24 h (S) and immunoblotted (IB) with indicated antibodies (Abs). The graph shows the percentage of autophagic (autophagosome puncta staining) MCF10A GFP-LC3 adhered or 24 h suspended cells. (B) Adhered (A) or suspended (S) MCF10A cells were treated or not treated with 5% Matrigel (MGel), and LC3 processing was detected by IB. (C) Adhered Fv2E-ΔNPERK MCF10A cell lines were transfected with GFP-LC3 plasmid and treated or not treated (control) with 100 pM AP10287 (AP) (dimerizing molecule) for 24 h, or adhered GFP-CL3 MCF10A cells were treated or not treated (control) with 100 nM salubrinal; cells were fixed and the percentage of autophagic cells was scored and quantified using fluorescence microscopy. The right panels show representative images of autophagic Fv2E-ΔNPERK (+AP) and GFP-LC3 (+Sal) MCF10A cells. Blue, 4′,6′-diamidino-2-phenylindole (DAPI); green, GFP-LC3. Scale bars are 5 μm. (D) Adhered Fv2E-ΔNPERK MCF10A cell line transfected with GFP-LC3 plasmid and treated with 100 pM or 2 nM AP or with vehicle [Et(Oh)] for 24 h were stained with EtBr, and the percentage of apoptotic cells was further measured by FACS. Right graph, population doubling during exponential growth (from day 2 to 8) of Fv2E-ΔNPERK MCF10A cells treated with vehicle (ethanol) or 100 pM or 2 nM AP. Cells were collected, and viability was determined using trypan blue exclusion. n.s., not significant. (E) Whole-cell lysates from Fv2E-ΔNPERK MCF10A cells treated or not treated with 100 pM AP and in combination with 0.1 mM leupeptin and 20 mM NH₄Cl as indicated were analyzed by IB for the indicated antigens. Leupeptin- and NH₄Cl-mediated inhibition of lysosomal degradation resulted in LC3-II accumulation (lane 3). Densitometric analysis (bottom panel) for LC3 flux was determined using Image J software (n = 3). (F) MCF10A cells were transfected with 5 μg cDNA encoding GST-BHMT (glutathione S-transferase fused to betaine homocysteine S methyltransferase 1). After 24 h, the cells were cultured in full growth medium, serum-free medium (6 h), or medium with AP20185 (100 pM) in the presence or absence of 10 nM bafilomycin A1 (BafA). Whole-cell lysates were immunoblotted for the indicated antigens. Myc Ab was used to detect GFP-myc (expressed from an internal ribosome entry site [IRES] sequence) as a control for transfection efficiency.