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. 2015 Oct 19;6(38):40704–40718. doi: 10.18632/oncotarget.5727

Figure 7. Myeloma recovery following UCH-L1 depletion is accompanied by the re-expression of UCH-L1.

Figure 7

A. KMS-11 cells transduced or not with dox-inducible UCHL1 targeting shRNA were cultured with doxycycline and cell viability was determined daily using the MTS assay. The graph represents the mean +/− SEM of triplicate cultures. Similar results were obtained in more than three independent experiments. B. The DUB inhibitor WP1130 induces cell death in shRNA-resistant KMS-11 cells. KMS-11 cells transduced with shRNA as in (A) were cultured for 15 days, and shRNA resistant cells were subsequently cultured with WP1130 (5 μM) where indicated. Cell viability was monitored after 48 hours as in (A) C. Schematic of the structure of the lentiviral shRNA construct. TRE = tetracycline responsive element, tRFP = turbo Red Fluorescent Protein. D. Samples from cells grown in (A) were collected on the indicated days and immunoblotted for the indicated proteins. E. Quantitative real-time PCR (qRT-PCR) was conducted on cells from (A) on the indicated days. The relative fold-change UCHL1 levels were determined using the delta-delta Ct method. F. Genomic DNA was isolated from cells in (A) on the indicated days. Following PCR amplification of a 3′-region of the UCHL1 gene, Sanger sequencing was performed on the region bound by the shRNA (chr4: 41270121-41) as indicated. The image represents chromatograms of two samples each for day 0 (pre-doxycycline) and on day 15 when cells had recovered.