FIGURE 10:
IRE1α modulates autophagosome volume. (A) COS-1 cells were transiently cotransfected with FLAG-IRE1α WT or mutants and mCherry-eGFP-LC3B. Cells were untreated or treated with chloroquine (CQ, 50 μM) and examined after 6 h. The mCherry-eGFP-LC3B reporter was not transfected in the cells shown in the first row (negative control). (B) Quantification revealed that the punctal area (the cross-sectional area occupied by autophagic puncta per 1000 μm2 of total cell area) was lower in chloroquine-treated K599A- and ΔR-overexpressing cells compared with treated WT or vector/control (*p = 1.11 × 10−10 [IRE1α]; p < 2.2 × 10−16 [CQ]; p = 4.19 × 10−8 [IRE1α × CQ interaction]). (C) Cross-sectional area per autophagosome (punctum size) paralleled punctal area. IRE1α WT or S724A increased punctum size compared with control, K599A, or ΔR (*p = 4.20 × 10−5 [IRE1α]; p < 2.2 × 10−16 [CQ]; p = 7.82 × 10−3 [IRE1α × CQ interaction]). (D) Cells expressing empty vector or FLAG-IRE1α WT or mutants were immunostained with anti-FLAG antibody (nonimmune IgG in controls). IRE1α WT. and mutants show cytoplasmic staining with perinuclear accentuation. For B and C, the number of trials and the number of quantified cells are indicated beneath columns.