Journal: Blood

Article Title: Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice

doi: 10.1182/blood-2011-11-394346

Figure Lengend Snippet: A-I06 suppresses the expression of functional XBP-1, and A-I06–treated B cells phenocopy B cells deleted with XBP-1 gene. (A) Structures and chemical synthesis of A-I06, A-I07, STF-083010, and B-A05, including the x-ray structure of STF-083010 with hydrogens omitted for clarity. (B) Assessment of STF-083010 aqueous stability using RP-HPLC. Injection of the freshly prepared DMSO stock solution of crystalline STF-083010 showed only minimal decomposition. STF-083010 subjected to multiple freeze-thaw cycles showed significant breakdown. Dissolution of STF-083010 in a 1:1 DMSO:water mixture resulted in complete conversion to A-I06 and A-I07 after 1 hour. Results shown here are representative of 3 independent experiments. (C) Wild-type B cells were stimulated with LPS (20 μg/mL) for 48 hours to allow the expression of XBP-1, and subsequently treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 24 hours. Cells were lysed and analyzed for the expression of XBP-1, IRE-1, calreticulin, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens. (D) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed and analyzed for the expression of XBP-1, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (E) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed in TRIzol reagent to extract RNA. Unspliced and spliced forms of mouse XBP-1 mRNA, and mouse actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (F) WaC3 cells were treated with DMSO (control), STF-083010 (50 μM), A-I06 (50μM), or A-I07 (50μM) for 72 hours, and subsequently lysed for RNA extraction. Unspliced and spliced forms of human XBP-1 mRNA, and human actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. (G) Wild-type B cells were cultured in the presence of LPS and A-I06 (50μM) for indicated times and lysed for analysis by immunoblots using Abs against μ heavy chain, p97 and actin. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens.

Article Snippet: Abs to TCL1 (Cell Signaling Technology), IRE-1 (Cell Signaling Technology), XBP-1 (Santa Cruz), Blimp-1 (Santa Cruz), IRF4 (Cell Signaling Technology), Pax5 (Santa Cruz), Syk (Cell Signaling Technology), phospho-Syk (Tyr525/526; Cell Signaling Technology), AKT (Cell Signaling Technology), phospho-AKT (Ser473; Invitrogen), ERK1/2 (Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology), GRP94 (Stressgen), calreticulin (Stressgen), calnexin (Stressgen), phospho-eIF2α (Ser51; Cell Signaling Technology), eIF2α (Cell Signaling Technology), HSP70 (Stressgen), p97 (Fitzgerald), CHOP (Cell Signaling Technology), actin (Sigma-Aldrich), activation-induced cytidine deaminase (AID; Cell Signaling Technology), phospho-Igα (Tyr182; Cell Signaling Technology), μ (SouthernBiotech), and κ (SouthernBiotech) were obtained commercially.

Techniques: Expressing, Functional Assay, Injection, Dissolution, Control, Western Blot, Purification, Isolation, Cell Culture, Reverse Transcription, RNA Extraction

Journal: Pacing and clinical electrophysiology : PACE

Article Title: Partially dominant mutant channel defect corresponding with intermediate LQT2 phenotype.

doi: 10.1111/j.1540-8159.2011.03222.x

Figure Lengend Snippet: Figure 4. Cell surface expression of G816V HERG compared to WT HERG. (A) Immunoblot of cell surface proteins that were isolated by biotin labeling and pull-down from transiently transfected HEK cells. Samples were separated by 7.5% linear SDS-PAGE. Calnexin was the negative control for surface labeling and cadherin was the positive control for surface labeling in the lower panels. Western blot was performed with anti-HERG, anti-calnexin, and anti-cadherin antibodies. (B) Summary data for part A. Densitometry analysis of the surface expression of HERG proteins was quantified as the amount of surface HERG divided by the total cellular HERG and also normalized for streptavidin pull-down of biotinylated protein (normalization calculated using cadherin and calnexin controls). WT HERG is normalized to 1.0. n = 4, *P < 0.05. (C) Confocal immunofluorescence micrographs of HEK 293 cells transfected with either WT HERG or G816V HERG and counter-stained with anti-cadherin antibody to indicate the cell membrane. Pearson correlation of HERG and cadherin colocalization is 0.571 ± 0.025 for WT HERG and 0.332 ± 0.029 for G816V, P < 0.001. n = 15, scale bar = 10 μm, DIC = differential interference contrast. (D) Confocal immunofluorescence micrographs of HEK 293 cells transfected with either HERG-WT or HERG-G816V and counter- stained with anti-calnexin antibody to indicate the ER, an intracellular compartment. Pearson correlation of HERG and calnexin colocalization is 0.831 ± 0.026 for WT HERG and 0.916 ± 0.006 for G816V, P < 0.01. n = 13, scale bar = 10 μm.

Article Snippet: Loading control antibodies used were pan-cadherin (Abcam), calnexin (Santa Cruz Biotechnology), Na-K-ATPase (Upstate, Millipore, Billerica, MA, USA), and tubulin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Isolation, Labeling, Transfection, SDS Page, Negative Control, Positive Control, Staining, Membrane

Journal: Pacing and clinical electrophysiology : PACE

Article Title: Partially dominant mutant channel defect corresponding with intermediate LQT2 phenotype.

doi: 10.1111/j.1540-8159.2011.03222.x

Figure Lengend Snippet: Figure 5. Interaction of WT HERG and G816V HERG subunits. (A) Co-immunoprecipitation and subsequent Western blot from transiently transfected HEK 293 cells shows 3× FLAG-tagged WT HERG detected from a pull-down of myc-tagged G816V HERG in three different mixed combinations with 100% WT HERG and 100% G816V HERG as controls, n = 3. Lower panel shows the reverse experiment where myc-tagged G816V HERG was detected in a pull-down of 3× FLAG-tagged WT HERG from the same combinations, n = 3. (B) Immuno-blots of cell surface biotinylation of 3× FLAG-tagged WT HERG and myc-tagged G816V HERG alone or in a 50/50 mix. Proteins were separated by 7.5% SDS-PAGE. Top panel shows a blot with anti-FLAG antibody with cadherin and calnexin as controls. Lower panel shows a blot with anti-Myc antibody with cadherin and calnexin as controls, n = 3.

Article Snippet: Loading control antibodies used were pan-cadherin (Abcam), calnexin (Santa Cruz Biotechnology), Na-K-ATPase (Upstate, Millipore, Billerica, MA, USA), and tubulin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Immunoprecipitation, Western Blot, Transfection, SDS Page

Journal: Biochimica et biophysica acta

Article Title: Enhanced ubiquitin-proteasome activity in calreticulin deficient cells: a compensatory mechanism for cell survival.

doi: 10.1016/j.bbamcr.2008.03.004

Figure Lengend Snippet: Fig. 4. (A) Changes in the proteasome activity following induction of ER stress. Chy- motrypsin like activity of the proteasome were measured by first treating the cells with 0.1 μM thapsigargin (TG) 1h, 1 μg/ml tunicamycin (TN) 3h, or 7 μM brefeldin A (BF) 3 h, followed by preparation of cell lysate and proteasome assay (as described in Materials and methods). The values are mean±SE of three separate experiments. ⁎ pb0.05 significantly different as compared to untreated cells. † pb0.05 significantly different as compared to corresponding wt cells. (B) Overexpression of ER chaperones, BiP or calnexin (CNX), in wt cells did not affect proteasome activity. Wt cells were transiently transfected with myc-BiP or myc-CNX after 48 h cells were lysed and proteasome activity was assayed. Western blot with anti-myc and actin shows the level of expres- sion of myc-BiP, myc-CNX and actin in the same cell lysate used to measure proteasome activity. Bar graphs are mean±SE of three separate experiments. ⁎ pb0.05 significantly different as compared to non-transfected cells.

Article Snippet: Next day, the media was replaced with Opti-MEMmedia (Invitrogen) and cellswere transfectedwith 80 pmol of calnexin siRNA or a control siRNA (siRNA-A, Santa Cruz Biotechnology) using Lipofectamine2000.

Techniques: Activity Assay, Over Expression, Transfection, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Enhanced ubiquitin-proteasome activity in calreticulin deficient cells: a compensatory mechanism for cell survival.

doi: 10.1016/j.bbamcr.2008.03.004

Figure Lengend Snippet: Fig. 6. Changes in proteasome activity upon reintroduction of calreticulin or its deletion mutants in crt−/−cells. (A) Western blot analysis with anti-calreticulin antibody of the wt, crt−/−cells and crt−/−cells transiently transfected with expression vectors encoding calreticulin (CRT-crt−/−) or calreticulin deletion and point mutants (CRTNP, CRTPC, CRTW244A, CRTNP-W244A and CRTPC-W244A). m represents the location of the calreticulin deletion mutants. (B) Proteasome activity in the crt−/−, vector transfected (V), CRT, CRTNP, CRTPC, CRTW244A and CRTNP-W244A. The levels of activity were compared relative to non-transfected wt and crt−/−cells. Bar graphs are mean±SE of 3–6 separate experiments. ⁎ pb0.05 significantly different as compared to the vector-transfected crt−/−cells. (C) Effect of calnexin (CNX) depletion on proteasome activity in wt and crt−/−cells. Cells were transfected with siRNA to calnexin or a control non-targeting siRNA (siRNA-A). 24 h after transfection, cells were lysed and proteasome activity was measured. Lower panel is a Western blot showing the level of cellular calnexin after transfection with calnexin-siRNA or control siRNA. ⁎ pb0.05 significantly different as compared to siRNA-A transfected crt−/−cells. (D) Effect of kifunensine on proteasome activity in wt and crt−/−cells. Kifunensine was added to the cell cultures at a final concentration of 5 μg/ml. 4 h later cells were lysed and proteasome activity was measured. The values are mean±SE of three separate experiments. ⁎ pb0.05 significantly different as compared to non-treated cells.

Article Snippet: Next day, the media was replaced with Opti-MEMmedia (Invitrogen) and cellswere transfectedwith 80 pmol of calnexin siRNA or a control siRNA (siRNA-A, Santa Cruz Biotechnology) using Lipofectamine2000.

Techniques: Activity Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, Concentration Assay