The TCL1 oncoprotein inhibits activation-induced cell death by impairing PKCθ and ERK pathways by Gilles Despouy, Marjorie Joiner, Emilie Le Toriellec, Robert Weil, and Marc Henri Stern Blood Volume 110(13):4406-4416 December 15, 2007 ©2007 by American Society of Hematology

TCL1 inhibits PMA-induced cell death and growth arrest. TCL1 inhibits PMA-induced cell death and growth arrest. (A) Viable cell number of Jurkat-C (gray line)– and Jurkat-TCL1 (black line)–treated (solid line) or untreated (dashed line) with PMA was determined at different times, as indicated in the figure, using a cell viability analyzer (Vi-cell XR; Beckman, Hialeah, FL). Data are expressed as mean (± SE) of duplicate samples. Three additional experiments gave similar results. (B) Photomicroscopy of Jurkat-C or Jurkat-TCL1, treated or untreated with PMA for 3 days. Two additional experiments gave similar results. (C) Apoptotic cell fraction of Jurkat-C (gray box) or Jurkat-TCL1 (black box) induced with PMA was determined by flow cytometry as the annexin V–FITC–positive/PI-negative percentage at different times, as indicated in the figure. Data are expressed as mean (± SE) of duplicates samples. Two additional experiments gave similar results. (D) Cell-cycle distribution (S phase) using flow cytometry after PI staining was determined at 24 hours for Jurkat-C (gray box) or Jurkat-TCL1 (black box) induced or not induced with PMA. Three additional experiments gave similar results. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology

TCL1 inhibits PKCθ activation. TCL1 inhibits PKCθ activation. (A) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 for different times, as indicated. Thr538-PKCθ phosphorylation (p-PKCθ, top) and TCL1 expression (bottom) were determined by Western blotting. Total PKCθ expression was used as a loading control (middle). A representative experiment of the 3 performed is shown. (B) Jurkat-C (gray line) and Jurkat-TCL1 (black line) were stimulated with PMA for 7 hours and CD69 expression was assessed by flow cytometry. A representative experiment of the 3 performed is shown. (C) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 for 10 minutes at different concentrations. ZAP70 phosphorylation (p-ZAP70) and TCL1 expression were determined by Western blotting. β-Actin expression was used as loading control. A representative experiment of the 2 performed is shown. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology

TCL1 inhibits NF-κB activation. TCL1 inhibits NF-κB activation. (A) Jurkat-C and Jurkat-TCL1 were stimulated with PMA or UCHT1 at different times indicated in the figure. IκBα (p-IκBα, top) and PKCθ (p-PKCθ, middle) phosphorylation was determined by Western blotting. β-Actin expression was used as a loading control (bottom). A representative experiment of the 3 performed is shown. (B) Band shift assay of nuclear extracts from Jurkat-C and Jurkat-TCL1. Cells were either untreated or stimulated for 30 and 60 minutes with PMA in presence or absence of Bay 11-7085 as indicated in the figure. A constitutive DNA binding of NF-κB with the same intensity in Jurkat-C and Jurkat-TCL1 is indicated by a gray arrow. Asterisk (*) indicates lane containing a 100-fold excess of cold KBF1 oligonucleotide competitor. (C) Jurkat-C (▒) and Jurkat-TCL1 (■) were seeded at 0.5 × 106 cells/mL, and treated (□) or untreated (plain box) with PMA (20 ng/mL) in presence or absence of Bay 11-7085 added 1 hour before and during the stimulation period at indicated concentrations. The viable cell number was determined at 48 hours, using a cell viability analyzer (Vi-cell XR; Beckman). Data are expressed as mean (± SE) of duplicates samples. One additional experiment gave similar results. (D) Photomicroscopy of Jurkat-C and Jurkat-TCL1, treated or untreated with PMA for 3 days in presence or absence of Bay 11-7085 at indicated concentrations. Two additional experiments gave similar results. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology

TCL1 inhibits ERK activation. TCL1 inhibits ERK activation. (A) Jurkat-C (gray box) and Jurkat-TCL1 (black box) were either treated or untreated with PMA in presence or absence of U0126, added 1 hour before and during the stimulation period. Cells were collected after 24 hours, stained with PI, and analyzed for cell cycle distribution (S phase) using flow cytometry. Three additional experiments gave similar results. Error bars represent SD. (B) Photomicroscopy of Jurkat-C and Jurkat-TCL1, treated or untreated with PMA for 3 days in presence or absence of U0126. Two additional experiments gave similar results. (C) Jurkat-C and Jurkat-TCL1 were stimulated with PMA at different times, as indicated in the figure. ERK phosphorylation (p-ERK) and TCL1 expression were determined by Western blotting. Total ERK expression was used as a loading control. A representative experiment of 5 performed is shown. Two polyclonal populations from independent transfections were tested with similar results (data not shown). (D) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 at different times, as indicated in the figure. ERK phosphorylation (p-ERK), AKT phosphorylation (p-AKT), and TCL1 expression were determined by Western blotting. β-Actin expression was used as a loading control. A representative experiment of 4 performed is shown. (E) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 for 10 minutes, and ERK was analyzed by immunofluorescence using an anti-ERK (FITC). Representative images of 3 independent experiments are shown. (F) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 in presence or absence of rottlerin (10 μM) or Gö6850 (1 μM), added 1 hour before and during the stimulation period. ERK phosphorylation was determined by Western blotting. After quantification, the p-ERK/β-actin ratio was determined as indicated at the bottom. Vertical lines have been inserted to indicate a repositioned gel lane. A representative experiment of 2 performed is shown. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology

TCL1 is the actor of cellular and biochemical phenotypes observed in Jurkat cells. TCL1 is the actor of cellular and biochemical phenotypes observed in Jurkat cells. (A) Jurkat-TCL1 cells were transduced with shRNA(GFP)-C and shRNA(GFP)-TCL1 vectors and stimulated with PMA (left) or UCHT1 (right) at the times indicated in the figure. ERK phosphorylation and TCL1 expression were determined by Western blotting. Total ERK expression was used as a loading control. A vertical line has been inserted to indicate a repositioned gel lane. A representative experiment of 2 performed is shown. (B) Photomicroscopy of Jurkat-TCL1 transduced with shRNA(GFP)-C and shRNA(GFP)-TCL1 vectors and treated or untreated with PMA for 3 days. (C) Jurkat cells were cotransfected with TCL1, MTCP1, or control vector and GFP-expression vector. Sorted GFP-positive transient transfected Jurkat cells (Jurkat-C*, Jurkat-TCL1*, and Jurkat-MTCP1*) were stimulated with PMA for 20 minutes (left). ERK phosphorylation and ERK expression were determined by Western blotting. After quantification, the p-ERK/total ERK ratio was determined as indicated at the bottom. (D) Transient transfected cells Jurkat-C* and Jurkat-TCL1* were stimulated with UCHT1. PKCθ phosphorylation and TCL1 expression were determined by Western blotting. Total PKCθ expression was used as a loading control. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology

Decreased TCL1 expression in SUP-T11 cell line restores PKCθ pathway activation. Decreased TCL1 expression in SUP-T11 cell line restores PKCθ pathway activation. (A) SUPT-11 cells were transduced with shRNA-C, shRNA-TCL1(1), and shRNA-TCL1(4) vectors and stimulated with PMA (10 ng/mL) at the times indicated in the figure. ERK phosphorylation and TCL1 expression were determined by Western blotting. Total ERK expression was used as loading control. A representative experiment of 3 performed is shown. After quantification, the p-ERK/ERK ratio (top panel, white box: t = 0 minute; gray box: t = 5 minutes; black box, t = 30 minutes of PMA induction) and TCL1 expression inhibition (bottom panel, a white box represents the mean plus or minus SE of the 3 time points for each shRNA) were determined and related to shRNA control and represented as histograms. A representative experiment of 2 performed is shown. (B) SUPT-11 cells were transduced with shRNA-C, shRNA-TCL1(1), and shRNA-TCL1(3) vectors and stimulated with UCHT1 at the times indicated in the figure. PKCθ phosphorylation and TCL1 expression were determined by Western blotting. β-Actin expression was used as loading control. After quantification, the pPKCθ/β-actin ratio (top panel, white box: t = 0 minute; gray box: t = 5 minutes; black box, t = 30 minutes of PMA induction) and TCL1 expression inhibition (bottom panel, a white box represents the mean plus or minus SE of the 3 time points for each shRNA) were determined and related to shRNA control and represented as histograms. A representative experiment of 2 performed is shown. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology

Expression of TCL1 in primary transformed and untransformed T lymphocytes inhibits PKCθ and ERK activation. Expression of TCL1 in primary transformed and untransformed T lymphocytes inhibits PKCθ and ERK activation. (A) Human normal PBMCs and T-PLL cells overexpressing TCL1 (TP22 on left and TP15 on right) were stimulated with PMA or UCHT1 at different times as indicated in the figure. PKCθ phosphorylation (top), ERK phosphorylation (2 exposures are shown to visualize the weaker signal in UCHT1 stimulation) (middle), and TCL1 expression (bottom) were determined by Western blotting. Total ERK and PKCθ expression were used as loading controls. A representative experiment of the 2 performed is shown. (B) PBMCs were transduced with pCDH1 (PBMC-(C) and pCDH1-TCL1 (PBMC-TCL1) vectors and stimulated with UCHT1 or PMA at the times indicated in the figure. ERK and PKCθ phosphorylation, and TCL1 expression were determined by Western blotting. Total ERK and β-actin expression were used as loading control. A representative experiment of 2 performed is shown. Gilles Despouy et al. Blood 2007;110:4406-4416 ©2007 by American Society of Hematology