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Blood, Vol. 111, Issue 3, 1644-1653, February 1, 2008
Protein kinase C–associated kinase is required for NF- B signaling and survival in diffuse large B-cell lymphoma cells
Blood Kim et al.
111: 1644
Supplemental materials for: Kim et al
Files in this Data Supplement:
- Figure S1. Analysis of infection rates of the shRNA-expressing lentiviruses (JPG, 72.5 KB)
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OCI-Ly10 cells were infected with the recombinant lentiviruses that express a control shRNA (L-shControl) or a PKK-specific shRNA (shPKK-1 or shPKK-2). Two days after infection, the fractions of the GFP-positive (infected) cells were analyzed by flow cytometry. Shown are data from three separate infections.
- Figure S2. PKK knockdown inhibits nuclear accumulation of NF-κB proteins (JPG, 66 KB)
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OCI-Ly10 cells were infected with the recombinant lentiviruses that express a control shRNA (L-shControl) or a PKK-specific shRNA (shPKK-1 or shPKK-2). Sixty-four hours post-infection, mRNA levels of PKK in cells expressing the indicated shRNA were analyzed by RT-PCR (A); and the levels of NF- B proteins in the nuclear and cytoplasmic fractions were assessed by western blot analysis (B).
- Figure S3. Suppression of PKK expression inhibits NF-κB activity in GCB DLBCL cell line OCI-Ly7 cells (JPG, 47.7 KB)
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OCI-Ly7 cells stably expressing the indicated shRNA were cultured in serum-free medium for two days. The expression levels of the indicated proteins were analyzed by western blotting.
- Figure S4. Suppression of PKK expression inhibits NF-κB activation induced by BAFF (JPG, 38.3 KB)
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(A) SUDHL-6 cells stably expressing shControl, shPKK-1 or shPKK-3 were cultured in serum-free medium overnight and then treated with BAFF (200 ng/ml) for 5 minutes. The expression levels of the indicated proteins were analyzed on western blots. (B) OCI-Ly7 cells stably expressing shControl or shPKK-1 were cultured in serum-free medium overnight and then treated with BAFF for 30 minutes. The expression of the indicated proteins was analyzed by western blotting.
- Figure S5. PKK is essential for the survival of ABC DLBCL cells (JPG, 52.2 KB)
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(A) OCI-Ly10 cells were infected with the lentiviruses expressing either L-shControl or L-shPKK-2. At the indicated times after infection, the GFP-positive fractions were analyzed by flow cytometry. The infection rate (GFP-positive cells), measured at 2 days post-infection, for shControl and shPKK-2 expressing viruses were 36% and 28%, respectively. The GFP-positive fractions were normalized to the samples harvested at 2 days post-infection. (B) OCI-Ly3 cells were infected with the lentiviruses expressing either L-shControl or L-shPKK-1. At the indicated times after infection, the GFP-positive fractions were analyzed by flow cytometry. The infection rate (GFP-positive cells), measured at 4 days post-infection, for shControl and shPKK-1 expressing viruses were 22.3% and 22.2%, respectively. The GFP-positive fractions were normalized to the samples harvested at 4 days post-infection.
- Figure S6. PKK is not required for the survival of OCI-Ly7 and SUDHL-6 cells cultured in medium containing 10% serum (JPG, 69.2 KB)
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OCI-Ly7 or SUDHL-6 cells were infected with the retroviruses (pRetro-H1) that express GFP together with either shControl or shPKK-1. The GFP positive fractions of OCI-Ly7 or SUDHL-6 cells stably expressing either shControl or shPKK-1 were analyzed by flow cytometry at the indicated times. The GFP-positive fractions were normalized to the samples harvested at day 1 of the analysis.
- Figure S7. Effect of PKK knockdown on survival of OCI-Ly7 cells (JPG, 55.8 KB)
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(A) OCI-Ly7 cells stably expressing shControl or shPKK-1 were cultured in the medium containing indicated concentrations of serum for 24 hours. The live cells were measured by the MTT assay. The results were normalized to the absorbance readings at the time the cells were plated (0 hr), which were arbitrarily set at 100.
- Figure S8. Effect of PKK knockdown on cell cycle progression of SUDHL-6 cells (JPG, 73.3 KB)
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SUDHL-6 cells stably expressing shControl or shPKK-1 were cultured in serum-free for the indicted times. BrdU was added into the culture medium for 1 hour before cells were harvested, and the cell cycle distribution of the indicated cells was analyzed as previously described.63 The percentage of S phase fraction of cells at different time points, as compared with the S phase fraction at 0 hr, which is set at 100%, is indicated below each FACS profile.
REFERENCES
Su C, Gao G, Schneider S, et al. DNA damage induces downregulation of histone gene expression through the G1 checkpoint pathway. EMBO J. 2004;23:1133-1143.
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