FIGURE 11:
IRE1α-mediated autophagosome formation does not require RNase activity. (A) COS-1 cells were treated with chloroquine (CQ, 50 μM), the IRE1α RNase inhibitor 4μ8c (10 μM), or vehicle (DMSO). Quantification of the rate of autophagosome formation for up to 24 h as reported by LC3B-II normalized to actin (C) or the LC3B-II to I ratio (D) revealed no significant differences in the presence or absence of 4μ8c, although LC3B-II increased over time (C: p = 2.21 × 10−6; D: p = 9.34 × 10−5; three experiments). (B) Tunicamycin (T, 10 μg/ml) induced Xbp1 splicing, and this effect was blocked by 4μ8c (4μ, 10 μM) at 6 and 24 h. NR, no–reverse transcriptase negative control; NT, no-template negative control; Tx, treatment.