Figure 3.
DDI2 KO impairs proteasome recovery after irreversible inhibition and sensitizes to the PI carfilzomib. (A) Western blot showing the absence of DDI2 expression in 4 distinct biallelic DDI2-KO clones (orange font) obtained from AMO1-VR cells. GAPDH was used as loading control. (B) Percentage of DDI2 WT (red bars) or DDI2 KO (blue bars) viable cells at baseline and after treatment with 20 nM carfilzomib for 48 hours. Results were obtained from 3 distinct DDI2 KO monoclones and averaged. Average of 3 independent experiments. (C) Schema of DDI2 and DDI2* add-backs showing N terminus FLAG tag and the ubiquitin-like (UBL) and retroviral-like protease (RVP) domains. The critical loss-of-function mutation D252N in the catalytic domain is also shown. (D) Western blot showing expression of DDI2 and DDI2* in WT and DDI2-KO cells based on FLAG (top panel) and DDI2 (middle panel) expression. The endogenous (En) DDI2 band is visible in DDI2 WT cells in contrast to exogenously expressed DDI2 (Ex). (E) Baseline chymotryptic-like (CT-L) proteasome activity in DDI2 WT (red bar), DDI2-KO monoclones (blue bar), DDI2-KO monoclones plus DDI2 add-back (green bar) and DDI2-KO monoclones plus DDI2* add-back (purple bar) AMO1-VR. Average of 3 independent experiments using 2 distinct DDI2-KO clones is shown. (F) DDI2 WT AMO1-VR cells (solid red line), DDI2-KO clones (solid blue line), and DDI2-KO clones expressing DDI2 (dashed green line) or DDI2* (dashed purple line) add-back were treated with carfilzomib for 1 hour to almost fully inhibit proteasome function (time 0). CT-L proteasome activity was assessed at the indicated time points after carfilzomib wash out. Average of 3 independent experiments with 2 distinct DDI2-KO clones is shown. (G) Relative percentage of living cells upon 24 hours of treatment with carfilzomib in DDI2 WT AMO1-VR cells and 3 distinct DDI2 KO AMO1-VR clones (KO1, KO2, and KO3) expressing DDI2 (green bars) or DDI2* (purple bars) add-backs or an empty vector (blue bars). Values were normalized against DDI2 WT cells expressing empty vector and are the average of 3 independent biological replicates. *P < .05, **P < .001, ****P < .0001. ns. not significant (P > .05).

DDI2 KO impairs proteasome recovery after irreversible inhibition and sensitizes to the PI carfilzomib. (A) Western blot showing the absence of DDI2 expression in 4 distinct biallelic DDI2-KO clones (orange font) obtained from AMO1-VR cells. GAPDH was used as loading control. (B) Percentage of DDI2 WT (red bars) or DDI2 KO (blue bars) viable cells at baseline and after treatment with 20 nM carfilzomib for 48 hours. Results were obtained from 3 distinct DDI2 KO monoclones and averaged. Average of 3 independent experiments. (C) Schema of DDI2 and DDI2* add-backs showing N terminus FLAG tag and the ubiquitin-like (UBL) and retroviral-like protease (RVP) domains. The critical loss-of-function mutation D252N in the catalytic domain is also shown. (D) Western blot showing expression of DDI2 and DDI2* in WT and DDI2-KO cells based on FLAG (top panel) and DDI2 (middle panel) expression. The endogenous (En) DDI2 band is visible in DDI2 WT cells in contrast to exogenously expressed DDI2 (Ex). (E) Baseline chymotryptic-like (CT-L) proteasome activity in DDI2 WT (red bar), DDI2-KO monoclones (blue bar), DDI2-KO monoclones plus DDI2 add-back (green bar) and DDI2-KO monoclones plus DDI2* add-back (purple bar) AMO1-VR. Average of 3 independent experiments using 2 distinct DDI2-KO clones is shown. (F) DDI2 WT AMO1-VR cells (solid red line), DDI2-KO clones (solid blue line), and DDI2-KO clones expressing DDI2 (dashed green line) or DDI2* (dashed purple line) add-back were treated with carfilzomib for 1 hour to almost fully inhibit proteasome function (time 0). CT-L proteasome activity was assessed at the indicated time points after carfilzomib wash out. Average of 3 independent experiments with 2 distinct DDI2-KO clones is shown. (G) Relative percentage of living cells upon 24 hours of treatment with carfilzomib in DDI2 WT AMO1-VR cells and 3 distinct DDI2 KO AMO1-VR clones (KO1, KO2, and KO3) expressing DDI2 (green bars) or DDI2* (purple bars) add-backs or an empty vector (blue bars). Values were normalized against DDI2 WT cells expressing empty vector and are the average of 3 independent biological replicates. *P < .05, **P < .001, ****P < .0001. ns. not significant (P > .05).

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