Figure 4.

XBP1u Is Increased upon Proteasome Inhibition and Forms ALIS Structures in Cells Regulated by USP14

(A) SH-SY5Y cells treated with MG132 (20 μM), chloroquine (50 μM), or DMSO (negative control) for 6 h were analyzed by immunoblotting for the presence of XBP1u detected by XBP1 antibody. Lower panel depicts densitometry histograms for the value of XBP1u normalized to actin.

(B) SH-SY5Y cells transfected with GFP-WT-USP14 or empty vector were treated with MG132 (20 μM), bortezomib (0.5 μM), or DMSO for 6 h (negative control) and analyzed for the presence of XBP1u. Immunodetection with anti-GFP and anti-actin served as transfection and loading controls, respectively. Lower panel shows XBP1u normalized to actin.

(C) SH-SY5Y cells treated with MG132 (20 μM) or DMSO for 6 h were fixed and stained with anti-XBP1u antibodies. Note presence of immunopositive aggregates, named aggresome-like induced structures (ALIS, green). Nuclei were stained by Hoechst dye (blue). Scale bar, 100 μm.

(D) USP14 shRNA and scramble shRNA lentivirus-infected SH-SY5Y cells were treated with MG132 (20 μM) or DMSO for 6 h. The lysates were analyzed by IB for XBP1 (XBP1u), actin, and USP14. Right panel depicts a histogram for the densitometry ratio of XBP1u normalized to actin.

n = 3, p value was calculated by one-way (A and D) or two-way ANOVA (B). *p ≤ 0.05; ***p ≤ 0.001, ****p ≤ 0.0001.