GABARAP is a determinant of apoptosis in growth-arrested chicken embryo fibroblasts

Abstract

Nutrient depletion triggers a series of adaptive processes as part of the unfolded protein response or UPR. These processes reduce stress to the endoplasmic reticulum by enhancing its protein folding capacity or ability to promote the degradation of dysfunctional proteins. Failure to restore ER homeostasis causes the activation of lethal pathways. The expression of a dominant negative mutant of C/EBPβ (Δ184‐C/EBPβ) alters this balance in chicken embryo fibroblasts (CEF). As a result, CEF display enhanced survival upon prolonged nutrient depletion. Starved Δ184‐C/EBPβ–expressing CEF display pronounced features of autophagy characterized by the appearance of large vesicles containing amorphous material, the formation of smaller double‐membrane vesicles (autophagosomes) and processing of LC3 and GABARAP. However, there were marked differences in the expression and processing of these proteins. In both normal and Δ184‐C/EBPβ expressing CEF, the lipidated form of LC3 (form II) accumulated during starvation but was detectable even when cells were actively dividing in complete medium. In contrast, GABARAP expression and lipidation were strongly stimulated in response to starvation. Inhibition of LC3 expression by RNA interference led to apoptosis in normal CEF even in the absence of starvation but stable and near complete repression of GABARAP was tolerated. Moreover, the inhibition of GABARAP enhanced CEF survival and abolished the expression of the pro‐apoptotic CHOP factor in conditions of starvation, suggesting a reduced level of ER stress. Therefore, GABARAP is a determinant of apoptosis in CEF subjected to prolonged nutrient depletion. J. Cell. Physiol. 230: 1475–1488, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…6); 26,165 however, even if the total amount of LC3 does increase, the magnitude of the response is generally less than that documented in yeast. It is worth noting that since the conjugated forms of the GABARAP subfamily members are usually undetectable without induction of autophagy in mammalian and other vertebrate cells, 166,167 these proteins might be more suitable than LC3 to study and quantify subtle changes in autophagy induction.…”

“…Antibody specificity can be assessed after complete inhibition of GABARAP (or any other Atg8 family protein) expression by RNA interference. 143,167 In general, we advise caution in choosing antibodies for western blotting and immunofluorescence experiments and in interpreting results based on stated affinities of antibodies unless these have been clearly determined. As with any western blot, proper methods of quantification must be used, which are, unfortunately, often not well disseminated; readers are referred to an excellent paper on this subject (see ref.…”

“…1279 In chicken embryo fibroblasts, nearly complete and general (i.e., in nearly all cells) protein downregulation can be observed within a few days after transfection of the shRNA retroviral vector. 167 Cautionary notes: Since the DT40 cell line derives from a chicken bursal lymphoma, not all ATG proteins and autophagy-regulatory proteins are detected by the commercially available antibodies produced against their mammalian orthologs; however, commercially available antibodies for mammalian LC3 and GABARAP have been reported to detect the chicken counterparts in western blots. 167 The chicken genome is almost completely assembled, which facilitates the design of targeting constructs.…”

Extracellular Signal-Regulated Kinase 2 and CHOP Restrict the Expression of the Growth Arrest-Specific p20K Lipocalin Gene to G0

“…However, this expression is transient and inhibited when cells are kept at confluence for several days without medium/nutrient replenishment. The downregulation of p20K is associated with induction of the CHOP transcriptional regulator, a marker of starvation and ER stress, and with a progressive decline in cell numbers (25,28) (Fig. 1A and data not shown).…”

“…Protein lysates were generated from confluent or subconfluent cultures by seeding CEF at a density of 1 ϫ 10 5 cells per 100-mm dish and preparing samples by cell lysis in SDS sample buffer at different times after seeding. The following primary antibodies and dilutions were used in these analyses: chicken p20K, 1:2,000 (Western blotting) and 1:300 (immunofluorescence assay) (19); chicken CHOP, 1:1,500 (25); chicken C/EBP␤, 1:1,500 (Western blotting) and 1:100 (immunoprecipitation) (21); ERK2, 1:2,000 (Western blotting) and 1:100 (immunofluorescence assay) (clone 1B3B9; Millipore); HIF1␣, 1:500 (ab2185; Abcam); and ␤-tubulin, 1:50,000 (T7816; Sigma-Aldrich, St. Louis, MO). Following several washes, the blots were incubated with a 1:25,000 dilution of a secondary anti-rabbit, anti-mouse, or anti-goat IgG antibody conjugated with horseradish peroxidase (HRP) at room temperature in Tris-buffered saline (TBS) with 5% milk.…”