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. 2019 Oct 10;8:e49796. doi: 10.7554/eLife.49796

Figure 3. Acute stressors destabilize mutant emerin protein levels.

(A) C2C12 cells stably expressing EMDΔ-GFP and treated with DMSO vehicle control, CHX alone, CHX and MG132, or MG132 alone for 8 hours. All images were acquired using the same laser power and detector gain settings. Single confocal z slices shown. (B) Western blot analysis of protein levels in C2C12 cells stably expressing EMDΔ-GFP and treated with DMSO vehicle, the translation inhibitor CHX, the proteasome inhibitor MG132, the p97 ATPase inhibitor eeyarestatin, or the glycosylation trimming inhibitor kifunensine for the time periods shown. a-tubulin shown as loading control. (C) Western blot analysis of U2OS cells stably expressing EMDΔ-GFP and doxycycline-inducible RNAi targeting the E2 ubiquitin ligases UBE2G1 and UBE2G2 and treated with DMSO vehicle control (-) or with doxycycline (+) for 48 hours. Free GFP indicates RNAi induction. a-tubulin shown as loading control. (D) Western blot detection of EMDΔ-GFP levels in cells treated with DMSO vehicle, or co-treated with CHX and the ER stress inducer THG for the time periods shown. a-tubulin shown as loading control. (E) C2C12 cells stably expressing EMDΔ-GFP and treated with vehicle control or with THG for the time periods shown. Insets show nuclei in the same ~50 µm field of view stained with Hoechst. All images acquired using the same laser power and detector gain settings; single confocal z slices shown. (F) Quantification of total NE-localized GFP fluorescence in maximum intensity projections of confocal z slices acquired across the conditions shown in (E) for N > 410 cells per condition. (G) Diagram of emerin domain organization and the sequence of an inserted C-terminal glycosylation sequence derived from the opsin protein, with glycosylation acceptor site marked (*). (H) Analysis of EMDΔ-GFP* glycosylation state in cells subjected to treatment with DMSO vehicle control or CHX and THG cotreatments for the times indicated. Red arrowhead indicates EndoH-sensitive glycosylated state of EMDΔ-GFP*; orange arrowhead indicates EndoH-resistant states of EMDΔ-GFP*; black arrowhead indicates deglycosylated EMDΔ-GFP*. a-tubulin shown as loading control. Numbers to left of blots indicate molecular weights in kDa. Scale bars in micrographs indicate 10 mm. See also Figure 3 – figure supplement 1, 2, and 3.

Figure 3.

Figure 3—figure supplement 1. Localization and stability of a disease-linked emerin variant.

Figure 3—figure supplement 1.

(A–B) C2C12 cells stably expressing EMDΔ-GFP (A) or EMD-WT-GFP (B) and treated with DMSO vehicle control, CHX alone, CHX and MG132, or MG132 alone for 8 hr. All images were acquired using the same laser power and detector gain settings. Single confocal z slices shown. Scale bar, 10 μm. (C–D) Western blot analysis of EMDΔ-GFP (C) or EMD-WT-GFP (D) protein levels after treatment with CHX for the indicates times.
Figure 3—figure supplement 2. siRNA-mediated E2 or E3 ubiquitin ligase knockdowns do not stabilize EMDΔ-GFP.

Figure 3—figure supplement 2.

(A–B) EMDΔ-GFP protein levels do not change in C2C12 cells stably expressing EMDΔ-GFP and transfected in duplicate with 50 nM RNAi targeting the E3 ubiquitin ligases Rnf26, CGRRF1, MARCH6, or a scrambled control (A), or targeting the E2 ubiquitin conjugating enzymes UBE2G1, UBE2G2, UBE2J1, and UBE2J2, or MARCH6 (B). (C) qPCR quantification of RNAi knockdown efficiency for conditions shown in (A–B). (D) EMDΔ-GFP protein levels either decrease or do not change in U2OS cells stably expressing EMDΔ-GFP and induced to express a miR-E RNAi cassette targeting the E3 ubiquitin ligase MARCH6 or the E2 ubiquitin conjugating enzyme UBE2J1 for 48 hr. Free GFP is also expressed from this cassette and indicates RNAi expression. miR-E RNAis used were from a validated set described in Knott et al. (2014).
Figure 3—figure supplement 3. Glycosylation reporter variants are destabilized by ER stress and recovered by BFA treatment.

Figure 3—figure supplement 3.

Glycosylation reporter variant of EMDΔ-GFP localizes normally to the NE and responds to ER stress induced by THG and secretory pathway disruption caused by BFA (A–B). (C) Pattern of glycosylation modifications in DMSO vehicle control treated cells or cells treated with THG for 2–4 hr. A larger proportion of EMDΔ-GFP becomes Endo H-resistant during acute ER stress.