Journal: The EMBO Journal

Article Title: hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

doi: 10.1038/sj.emboj.7601450

Figure Lengend Snippet: hRpn13 promotes isopeptidase activity of UCH37. (A) UCH37 binds to S5a in vitro. Purified GST or GST-fused UCH37 was incubated with purified recombinant His-tagged S5a for 1 h. Pull-down assays using GSH beads were then performed. The protein levels were analyzed by Western blot. (B) Recombinant hRpn13 stimulates the ubiquitin-amc (Ub-amc) hydrolysis by UCH37. In the presence of 0.5 μM of Ub-amc, 0.1 nM of UCH37 was incubated with 0 or 10 nM of the full-length hRpn13 (FL, from bacteria), the C-terminal half of hRpn13 (Δ1–200, from bacteria), or the full-length hRpn13 expressed in insect cells (FL-ins). Ub-amc hydrolysis (in arbitrary units) was determined during incubation for 30 min by monitoring the release of amc. (C) Unlike hRpn13, pure S5a/Rpn10 cannot promote the isopeptidase activity of UCH37 in vitro. UCH37 at indicated concentrations, was incubated with 0 (circle) or 10 nM of hRpn13 (square, expressed in insect cells), S5a (cross), or hRpn13 plus 10 nM of Ub-aldehyde (triangle). Ub-amc hydrolysis (in arbitrary units) was determined as in (B). (D) hRpn13 does not increase the isopeptidase activity of the 26S proteasome or UCHL3. Ub-amc was incubated (as in 5B) without (control) or with 10 nM of purified hRpn13 in the presence of UCH37 (0.1 nM), the 26S proteasome (0.24 μg/ml, about 0.1 nM), or UCHL3 (0.01 nM). Ub-amc hydrolysis (in arbitrary units) was determined as in (B). (E) hRpn13 decreases the levels of ubiquitin conjugates in cells. Left, 293T cells were transfected with an empty vector or Myc/His6-tagged hRpn13. Right, 293T cells were transfected with pBS/U6/GFP (GFP siRNA) or pBS/U6/hRpn13 (hRpn13 siRNA). The cells were incubated for 2 days after transfection, and the contents of hRpn13, β-actin, and large ubiquitin conjugates (molecular weights >191 kDa) were assayed by Western blot.

Article Snippet: In addition, a pool of four siRNA oligos specific for hRpn13 (Smartpool) and a nontargeting siRNA oligo were purchased (Dharmacon Inc.) and transfected using trans-IT-TKO transfection kit (Mirus Inc.).

Techniques: Activity Assay, In Vitro, Purification, Incubation, Recombinant, Western Blot, Ubiquitin Proteomics, Bacteria, Control, Transfection, Plasmid Preparation

Journal: The EMBO Journal

Article Title: hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

doi: 10.1038/sj.emboj.7601450

Figure Lengend Snippet: hRpn13 content influences rates of degradation of short-lived proteins in 293T cells. (A) Overexpression of hRpn13 or its C-terminal region reduces the degradation of short-lived proteins. 293T cells were transfected with an empty vector, hRpn13 or its C-terminal region (hRpn13-Δ1–200). After 2 days, the rate of degradation of cellular proteins was determined by pulse-chase analysis using [3H]tyrosine. A similar small reduction in degradation was seen in at least three experiments. (B) Decreasing the levels of hRpn13 also slows the degradation of short-lived proteins. 293T cells were transfected with pBS/U6/GFP (GFP siRNA) or pBS/U6/hRpn13 (hRpn13 siRNA). Then, degradation of short-lived proteins was determined as in (A). (C) Overexpression or knockdown of hRpn13 reduces the degradation of the model N-end rule substrate, Ub-R-GFP. Left, 293T cells were cotransfected with Ub-R-GFP, GFP, and hRpn13 (or empty vector). Right, 293T cells were cotransfected with Ub-R-GFP, GFP, and siRNA oligos (100 nM) for hRpn13 (or nontargeting siRNA oligos). The cells were incubated for 3 days after transfection, and the protein levels were analyzed by Western blot. (D) Degradation of the natural proteasomal substrate, ErbB3, is reduced by overexpression of hRpn13 or its C-terminal half. 293T cells were cotransfected with ErbB3, EGFR, and hRpn13 (or hRpn13-Δ1–200 or empty vector), and incubated for 2 days after transfection. Protein levels were analyzed by Western blot.

Article Snippet: In addition, a pool of four siRNA oligos specific for hRpn13 (Smartpool) and a nontargeting siRNA oligo were purchased (Dharmacon Inc.) and transfected using trans-IT-TKO transfection kit (Mirus Inc.).

Techniques: Over Expression, Transfection, Plasmid Preparation, Pulse Chase, Knockdown, Incubation, Western Blot

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: p38 Inhibitor SCIO-469 can reverse TNF-mediated myelosuppression. MDS1 cells were pretreated for 1 hour with vehicle (–) or 1.0 μM SCIO-469 (+) and then induced with either 1 ng/mL TNFα or 5 ng/mL TGFβ for 30 minutes. The p-p38 and total p38 levels were analyzed by Western blotting. Bar graph represents p-p38 levels relative to total p38 in each sample (A). Immunomagnetically selected bone marrow–derived CD34+ cells were differentiated into hematopoietic progenitors at the CFU-E stage of maturation as described before.30 These cells were treated with 20 ng/mL TNFα or 10 000U/mL IFN-γ in the presence and absence of 100 nM SCIO-469. Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an antibody against the phosphorylated form of MapKapK-2 (threonine 334). The same blot was stripped and reprobed with an antibody against total MapKapK-2, to control for protein loading (B). Primary bone marrow–derived CD34+ cells were grown in cytokine-enriched liquid media in the presence and absence of 20 ng/mL TNFα and SCIO-469 (100 nM) for 24 hours. The percentages of apoptotic and dead cells were determined by staining with mixture of Annexin V–Alexa Fluor 488 and nucleic acid dye, Sytox green, respectively (Vybrant Apoptosis Kit; Molecular Probes) (C). Mean of 3 independent experiments showed significant decrease in TNFα-mediated apoptosis in the presence of SCIO-469 (P = .01, paired t test). BM CD34+ progenitors were cultured with TPO, Flt3L, and SCF with or without 20 ng/mL TNFα and in the presence and absence of 500 nM SCIO-469 for 6 days. BrDU incorporation was evaluated against the amount of 7-AAD by flow cytometry to determine the percentage of subpopulation at each cell-cycle stage in a gated population of CD34+ cells. Results from 3 experiments were used to compare the proportion of cells in G0/G1 and S phase of cell cycle by using 2-tailed t test. (D) Primary bone marrow–derived CD34+ cells were cultured in methylcellulose in the presence and absence of 20 ng/mL TNFα and SCIO-469. Colonies were scored on day 14. Results are expressed as mean ± SEM of 3 independent experiments (E). Treatment with SCIO-469 led to a significant reversal of TNF-mediated myelosuppression (P = .04, t test).

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Western Blot, Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Control, Staining, Cell Culture, BrdU Incorporation Assay, Flow Cytometry

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: p38 inhibitor SCIO-469 can decrease apoptosis in MDS CD34+ progenitors. BM mononuclear cells from patients with MDS were cultured in the presence and absence of 500 nM SCIO-469 for 48 hours. Apoptosis in gated population of CD34+ cells was determined by Annexin V staining. Comparison of dot plots from 5 independent experiments shows a decrease in the percentage of Annexin V–positive CD34+ cells in samples treated with SCIO-469 (A). MDS CD34+ progenitors from 5 patients show significantly greater viability and decreased apoptosis after 48 hours of treatment with SCIO-469 (paired 2-tailed t test). Results are presented as means ± SEMs (B).

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Cell Culture, Staining, Comparison

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: Down-regulation of p38α by siRNA can stimulate hematopoiesis in MDS CD34+ progenitors. A mixture of 4 siRNAs against p38α were transfected in primary CD34+ hematopoietic progenitors using Mirus TKO transfection reagent. Western blotting showed a specific and significant decrease in total p38 protein levels (A). High transfection efficiency was shown by using fluorescent-labeled siRNAs (B). MDS CD34+ cells were transfected with either anti-p38α or control-scrambled siRNAs and grown in vitro in methylcellulose with cytokines. Colonies were scored on day 14, and results were expressed as means ± SEMs of 3 independent experiments. Significantly higher number of both myeloid (CFU-GM) and erythroid (BFU-E) colonies were observed in cells transfected with anti-p38 siRNAs (C). MDS CD34+ cells transfected with anti-p38α and scrambled control siRNAs were evaluated after 48 hours by Annexin V staining. Flow cytometry revealed a significantly higher percentage of viable cells (P = .045, t test) and fewer number of apoptotic cells (P = .04, t test) when transfected with anti-p38α siRNA (D). Results are presented as means ± SEMs of 5 independent experiments.

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Transfection, Western Blot, Labeling, Control, In Vitro, Staining, Flow Cytometry

Journal:

Article Title: Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

doi: 10.1182/blood-2006-05-023093

Figure Lengend Snippet: Pharmacologic p38 inhibitors stimulate hematopoiesis in MDS CD34+ progenitors. MDS bone marrow–derived CD34+ cells from 19 patients were plated in methylcellulose in the presence and absence of p38 inhibitors SB203580 (5 μM and 10 μM), SD-282, and SCIO-469; inactive structural analog SB202474 (10 μM); and Mek-1 inhibitor PD98059 (10 μM). Colonies were scored at day 14, and results were expressed as means ± SEMs of 19 independent experiments.

Article Snippet: The siRNA duplexes were labeled with FITC to show successful transfection in primary CD34 + cells (Label IT siRNA Tracker; Mirus, Madison, WI).

Techniques: Derivative Assay