Journal: eLife

Article Title: Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

doi: 10.7554/eLife.91678

Figure Lengend Snippet: ( a ) Expression of DDI2 in the CRISPR-generated clones of HAP1 cells used in this work was analyzed by western blot. ( b ) The experimental setup used in this study. Cells were pulse treated with bortezomib (Btz) or carfilzomib (Cfz) for 1 hr, then cultured in drug-free media for times indicated and analyzed as described. ( c ) The viability of wt- and DDI2 KO clones of HAP1 cells was measured using CellTiter-Glo, and the inhibition of β5 sites was measured with the Proteasome-Glo assay at times indicated; n=2–5. ( d ) Knockout of DDI2 inhibits the Nrf1 processing. Western blots of Btz-treated HAP1 cells. The sample in the first lane is wt cells treated with VCP/p97 inhibitor CB-5083 immediately after removal of Btz. VCP inhibitors blocks Nrf1 processing ( ; ; ). ( e ) MDA-MB-231 and SUM149 cells were analyzed by western blot 72 hr after transfection with DDI2 siRNAs ( f ) Theβ5 activity in siRNA-transfected SUM149 and MDA-MB-231 was measured using Suc-LLVY-AMC immediately and 18 hr after treatment with 100 nM Btz; n=3. ( g ) β5 activity was measured in HCT-116 cells with the Proteasome-Glo assay immediately and 18 hr after treatment with PIs; n=2. Figure 1—source data 1. PDF file containing and original full-size western blot membranes (anti-DDI2, anti-GAPDH) with molecular weight markers. Figure 1—source data 2. Excel file containing data for . Figure 1—source data 3. PDF file containing and original full-size western blot membranes (anti-Nrf1, anti-DDI2, anti-β-actin) with molecular weight markers. Figure 1—source data 4. PDF file containing and full-size western blot membranes (anti-DDI2, anti-β-actin). Additional lanes demonstrate that the knockdown of DDI2 is maintained throughout the experiment. Figure 1—source data 5. Excel file containing data and statistical analysis for . Figure 1—source data 6. Excel file containing data for .

Article Snippet: Cell line ( Homo sapiens ) , SUM149 , BioIVT , RRID: CVCL_3422 , .

Techniques: Expressing, CRISPR, Generated, Clone Assay, Western Blot, Cell Culture, Inhibition, Glo Assay, Knock-Out, Transfection, Activity Assay, Molecular Weight, Knockdown

Journal: eLife

Article Title: Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

doi: 10.7554/eLife.91678

Figure Lengend Snippet: ( a ) Cells were treated with PIs for 1 hr, media was shaken off, and cells were cultured in an inhibitor-free fresh media for 48 hr when Alamar Blue assay was performed; n=3–4. See Figure 1 in for a comparison of SUM149 and MDA-MB-231 cells. ( b ) The β5 proteasome activity was measured using Suc-LLVY-AMC in the cell extracts of untreated cells; n=2-8. Figure 1—figure supplement 2—source data 1. Excel file containing data for both panels.

Article Snippet: Cell line ( Homo sapiens ) , SUM149 , BioIVT , RRID: CVCL_3422 , .

Techniques: Cell Culture, Alamar Blue Assay, Comparison, Activity Assay

Journal: eLife

Article Title: Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

doi: 10.7554/eLife.91678

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , SUM149 , BioIVT , RRID: CVCL_3422 , .

Techniques: Generated, CRISPR, Mutagenesis, Transfection, Construct, Sequencing, Bradford Assay, Isolation, Reverse Transcription, SYBR Green Assay, Viability Assay, Software