Skip to main content
. 2022 May 10;11:e73913. doi: 10.7554/eLife.73913

Figure 4. DAC (S)–3 triggers ER-stress, ubiquitin-proteasome system inhibition and apoptosis.

(A) U2OS stably co-expressing a GFP variant addressed and retained in the endoplasmic reticulum were treated with 1 µM (S)–3 and monitored by live imaging. Representative pictures of U2OS cells, either untreated (NT) or treated with 1 µM (S)–3 for the indicated time. (B) Representative pictures of U2OS with stably GFP-labeled endoplasmic reticulum and either untreated or treated for 8 h with 1 µM (S)–3. (C) Immunoblotting of extracts from U2OS untreated or treated with 1 µM (S)–3 or 20 µM MG132 for 8 h. (D) Immunoblotting of ubiquitin in extracts from U2OS untreated or treated with 1 µM (S)–3 or 20 µM MG132 for 2 h. High-molecular-weight ubiquitin conjugates are indicated by a vertical bar on the right. (E) Representative pictures of U2OS stably expressing Ub-G76V-YFP and either untreated or treated for 4 h with 1 µM (S)–3 or 20 µM MG132. (F) Analysis of YFP fluorescence by flow cytometry of U2OS Ub-G76V-YFP treated as described in (E) ,% of cells scored as positives using the vertical green bar as a threshold are indicated. (G) Immunoblotting using extracts from U2OS cells treated with 1 µM (S)–3 for increasing times, indicated in hours. To probe for all the UPR markers, the same extracts were analyzed on different immunoblots, each one with its loading control (see the source data), which were grouped in logical order on the figure to facilitate the interpretation. (H) Immunoblotting using extracts from U2OS cells treated with 1 µM (S)–3 for 12 h with or without 50 µM z-VAD-fmk.

Figure 4—source data 1. Source data related to Figure 4C.
The tiff files correspond to uncropped pictures of the IRDye800CW fluorescence signal acquired on a LI-COR Odyssey. The regions used to generate the figure are highlighted by back squares in the jpg file.
Figure 4—source data 2. Source data related to Figure 4D.
The tiff files correspond to an uncropped picture of the IRDye800CW fluorescence signal, acquired on an Odyssey LI-COR, and of a scan of the membrane stained with Ponceau S. The regions used to generate the figure are highlighted by back squares in the jpg file.
Figure 4—source data 3. Source data related to Figure 4G.
The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc, of the IRDye800CW fluorescence signal acquired on a LI-COR Odyssey or of the chemiluminescent signal acquired using autoradiographic films. Several immunoblotting, labeled from a to f, of the same extracts were used to generate this figure, all with a loading control. The regions used to generate the figure are highlighted for each immunoblot by back squares in the jpg files.
Figure 4—source data 4. Source data related to Figure 4H.
The tiff files correspond to uncropped pictures of the IRDye800CW fluorescence signal acquired on a LI-COR Odyssey. The regions used to generate the figure are highlighted by back squares in the jpg file.

Figure 4.

Figure 4—figure supplement 1. DAC (S)–3 triggers ER-stress, inhibition of the Ub-proteasome system and apoptosis.

Figure 4—figure supplement 1.

(A) Representative micrographs of U2OS cells treated with 1 µM (S)–3 or 20 µM proteasome inhibitor MG-132 for 12 h. The black scale bar represents 10 µm. (B) Representative micrographs of U2OS cells treated for 12 h with 1 µM (S)–3 without or with 20 µg/ml translation inhibitor cycloheximide (CHX) which was pre-incubated 1 h with the cells before addition of (S)–3. The black scale bar represents 10 µm. (C) Analysis by immunoblotting of extracts from U2OS cells 48 or 72 h after transfection by control siRNA (Ctrl) or siRNA targeting PSMD2. (D) Representative micrographs of U2OS cells treated or not for 2 h with 1 µM (S)–3 or 20 µM MG132 and irradiated or not with 3 Gy of X-rays, post-incubated for 30 min, fixed and stained for the DNA damage markers gammaH2AX and the 53BP1 protein. Assembly of 53BP1 into foci relies on active ubiquitination at sites of DNA damage. The white scale bar represents 10 µm. (E) Cell viability analysis of U2OS cells pre-treated for 1 hr with 50 µM STF-083010 (IRE1α endonuclease inhibitor), 1 µM KIRA6 (IRE1α kinase inhibitor) or 0.5 µM GSK-2606414 (PERK inhibitor) before being co-treated with (S)–3 for 72 h. (F) Cell viability analysis of U2OS cells pre-treated for 1 h with 10 µM SP600125 (JNK inhibitor) before being co-treated with (S)–3. (G) Representative micrographs of U2OS cells treated or not with 1 µM (S)–3 with or without 50 µM z-VAD-fmk for 12 h. The black scale bar represents 10 µm.
Figure 4—figure supplement 1—source data 1. Source data related to Figure 4—figure supplement 1C.
The tiff files correspond to uncropped pictures of the IRDye800CW fluorescence signal acquired on a LI-COR Odyssey. The regions used to generate the figure are highlighted by back squares in the jpg file.