Figure 2.

GAB1 increases CLL cell homing capacity. (A) Representative immunoblot for MEC1^WT and MEC1 cells genetically engineered for GAB1 KO, using CRISPR/Cas9 (MEC1^GAB1-KO) cells (top); primary CLL cells transfected with siRNA against GAB1 (siGAB1) or with the control (siNC; bottom), cells were harvested after 48 hours posttransfection. (B) In vivo competitive migration assay of MEC1^WT vs MEC1^GAB1-KO. Cells were stained with CFSE or FarRed CellTrace dye, and incubated overnight. The next day, an equal number of CFSE- and FarRed–stained cells (each 50 × 10⁶) were mixed in 1:1 ratio (validated by flow cytometry), and injected into the NSG mice via the tail vein. Organ infiltration was analyzed by gating on human CFSE/FarRed⁺ cells. The plot depicts the CFSE/FarRed⁺ MEC1 cells present in peripheral blood of NSG mice. (C) Mice (n = 9) from the experiment described in panel B were euthanized 6 hours after cell transplantation, and blood, spleen, bone marrow, and liver were analyzed by flow cytometry to detect the presence of viable CFSE⁺ or FarRed⁺ cells. The amount of MEC1 cells in each examined organ site is presented as relative migration of CFSE⁺ vs FarRed⁺ cells. (D) An in vivo competitive migration assay of primary CLL cells (n = 5) transfected with siRNA against GAB1 (siGAB1) or negative control (siNC). After 48 hours, cells were stained with Sytox Blue and sorted for viable cells, and 10 × 10⁶ of cells from each condition were processed as described in panel B. In vivo migration continued for 4 hours, and blood, spleen, bone marrow, and liver were analyzed by flow cytometry to detect the presence of viable CFSE⁺ or FarRed⁺ cells. A small aliquot of sorted cells was assayed by immunoblot to confirm GAB1 silencing (supplemental Figure 9). (E) Representative immunoblots of MEC1 cells transfected with siRNA against GAB1 (siGAB1) or the control (siNC). After 48 hours, the cells were treated for 10 minutes with SDF1 (250 ng/mL), CXCL13 (500 ng/mL), or CM (100%) produced by primary human mesenchymal stromal cells (CM^hMSC). (F) An in vitro competitive migration assay of MEC1^WT vs MEC1^GAB1-KO cells (n = 4) stained as described in panel B. Migration toward SDF1 (250 ng/mL), CXCL13 (500 ng/mL), and CM^hMSC continued for 6 hours. (G) Representative immunoblots of primary CLL cells treated for 5 minutes with SDF1 (100 ng/mL), CXCL13 (250 ng/mL), or CM^hMSC. The immunoblot contained 2 endogenous controls (GAPDH) marked by the index at top, because for technical reasons, pGAB1^Y627, tGAB1, tAKT, and tERK (loading control GAPDH¹) were analyzed on the first gel and the remaining proteins (loading control GAPDH²) on the second gel (identical protein loading and conditions). (H) In vitro competitive migration assay of primary CLL cells transfected by siRNA against GAB1 (siGAB1) or control siRNA (siNC). Cells were stained as described in panel B and loaded onto a Transwell 48 hours after transfection. Migration continued for 6 hours toward SDF1 (100 ng/mL), CXCL13 (250 ng/mL), or CM^hMSC (n = 10 for SDF1 and CXCL13; n = 5 for CM^hMSC).