Figure 4.
GAB1 increases tonic AKT phosphorylation. (A) Representative immunoblots of primary CLL cells transfected with siRNA against GAB1 (siGAB1) or the negative control (siNC). Cells were harvested after 48 hours, divided into equal portions, and exposed to vehicle (control samples) or anti-IgM for 5 minutes (10 µg/mL). *pAKT (S473) is the same immunoblot as pAKT but was developed for a prolonged time. (B) Representative immunoblot of MEC1 cells transfected with a plasmid for GAB1 protein overexpression tagged with MYC sequence (GAB1MYC, top band) or with an empty vector (empty), and cultured for 48 hours. *β-Actin was the endogenous control for p-p85 and t-p85 assayed on a separate gel with identical loading. (C) Representative immunoblot of MEC1 cells transfected with siRNA against GAB1 (siGAB1) or the negative control (siNC) and harvested 48 hours after transfection. *β-Actin, as in panel B. (D) Representative immunoblot of MEC1 and OSU-CLL cells (CLL-derived cell line) with downmodulated GAB1 levels. MEC1 and OSU-CLL cells were infected with a lentiviral construct (permanent cell line) for tetracycline-inducible shRNA against GAB1 (shGAB1). shRNA expression was induced by adding tetracycline to medium (tet+) for 48 hours, and control cells were treated with vehicle (tet−). (Ei) Effect of GAB1 KO on MEC1 cell growth in vivo. MEC1WT and MEC1GAB1-KO cells were transduced with vectors expressing GFP or Azurite, mixed 1:1, and injected via tail vein into NSG mice (n = 3). Mice were euthanized 3 weeks after injection, and organs were analyzed by flow cytometry for the presence of MEC1WT and MEC1GAB1-KO cells labeled by GFP or Azurite in blood, spleen, bone marrow, and liver. We also performed a switch of fluorescent labels to exclude any potential effect of a specific fluorescent proteins on the cells (not shown). (Eii) Representative images from fluorescence microscopy of the spleen, bone marrow, and liver of NSG transplant recipients, as described in panel Ei (original magnification, ×20). (Fi) Representative immunoblot for the effect of FoxO1 attenuation by KO in MEC1 cells (MEC1WT vs MEC1FoxO1-KO cells) or by siRNA in primary CLL cells. Primary CLL cells were transfected with siRNA against FoxO1 (siFoxO1) or with control (siNC) and analyzed by immunoblot 48 hours later. (Fii) Representative immunoblots for MEC1 cells and primary CLL cells treated with FoxO1 inhibitor (inhFoxO1; 0.5 µM, 24 hours).

GAB1 increases tonic AKT phosphorylation. (A) Representative immunoblots of primary CLL cells transfected with siRNA against GAB1 (siGAB1) or the negative control (siNC). Cells were harvested after 48 hours, divided into equal portions, and exposed to vehicle (control samples) or anti-IgM for 5 minutes (10 µg/mL). *pAKT (S473) is the same immunoblot as pAKT but was developed for a prolonged time. (B) Representative immunoblot of MEC1 cells transfected with a plasmid for GAB1 protein overexpression tagged with MYC sequence (GAB1MYC, top band) or with an empty vector (empty), and cultured for 48 hours. *β-Actin was the endogenous control for p-p85 and t-p85 assayed on a separate gel with identical loading. (C) Representative immunoblot of MEC1 cells transfected with siRNA against GAB1 (siGAB1) or the negative control (siNC) and harvested 48 hours after transfection. *β-Actin, as in panel B. (D) Representative immunoblot of MEC1 and OSU-CLL cells (CLL-derived cell line) with downmodulated GAB1 levels. MEC1 and OSU-CLL cells were infected with a lentiviral construct (permanent cell line) for tetracycline-inducible shRNA against GAB1 (shGAB1). shRNA expression was induced by adding tetracycline to medium (tet+) for 48 hours, and control cells were treated with vehicle (tet−). (Ei) Effect of GAB1 KO on MEC1 cell growth in vivo. MEC1WT and MEC1GAB1-KO cells were transduced with vectors expressing GFP or Azurite, mixed 1:1, and injected via tail vein into NSG mice (n = 3). Mice were euthanized 3 weeks after injection, and organs were analyzed by flow cytometry for the presence of MEC1WT and MEC1GAB1-KO cells labeled by GFP or Azurite in blood, spleen, bone marrow, and liver. We also performed a switch of fluorescent labels to exclude any potential effect of a specific fluorescent proteins on the cells (not shown). (Eii) Representative images from fluorescence microscopy of the spleen, bone marrow, and liver of NSG transplant recipients, as described in panel Ei (original magnification, ×20). (Fi) Representative immunoblot for the effect of FoxO1 attenuation by KO in MEC1 cells (MEC1WT vs MEC1FoxO1-KO cells) or by siRNA in primary CLL cells. Primary CLL cells were transfected with siRNA against FoxO1 (siFoxO1) or with control (siNC) and analyzed by immunoblot 48 hours later. (Fii) Representative immunoblots for MEC1 cells and primary CLL cells treated with FoxO1 inhibitor (inhFoxO1; 0.5 µM, 24 hours).

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