SOX11 expression promotes pseudoemperipolesis through CXCR4 and FAK pathway activation in MCL. First we analyzed SOX11-dependent MCL cell migration beneath SNKT cells (pseudoemperipolesis). (A) SOX11⁺ and SOX11⁻ in vitro MCL cell line models untreated (Ø) or treated with PF or AMD were added to SNKT-GFP⁺ layers or (B) SOX11⁺ MCL cells derived from PB (>95% purified) of primary MCLs (n = 5) untreated (Ø) or pretreated with PF or AMD and untreated cells derived from SOX11⁻ (n = 4) MCL primary samples were cocultured with SNKT-GFP⁺ cells. After overnight incubations, cocultures described in panels A and B were washed several times, and MCL cells that had migrated toward the stromal layer were trypsinized and counted by FC; MCL cells were distinguished by GFP⁻ gating and cell size. Results in panel A are shown as relative to the corresponding untreated SOX11⁺ MCL cell line (Z138CTØ and JVM2SOX11Ø, respectively). (C) Bar graph representing the mean fluorescence of FC experiments showing the expression levels of cellular surface CXCR4 protein levels in Granta519 and Z138CT vs their KD MCL cell lines counterparts, and JVM2SOX11⁺ vs JVM2CT MCL cell lines growing alone (−CC) or in coculture with the human BM stromal-cell line (SNKT; +CC). Granta519 was used as a CXCR4⁻ control cell line. Isotype immunoglobulin G control antibody was used as a negative CXCR4 control staining. (D) Western blot experiments showing expression levels of basal forms of FAK, AKT, and ERK1/2 proteins and p-FAK, p-AKT, and p-ERK in Z138CT and Z138KD pretreated with PBS (−) or with PF (+) and cultured 30 minutes in RPMI plus 10% fetal bovine serum with PBS (−) or with CXCL12 (+). Actin was used as a loading control. (E) FAK Tyr³⁹⁷ phosphorylation was determined by confocal microscopy. Z138CT and Z138KD cells untreated (Ø) or pretreated with PF (+PF) were seeded over covered glasses growing alone (−CC) or in coculture with SNKT-GFP⁺ layers (green cells; +CC). After overnight incubation, nonadhered cells were removed by several washes. Adhered cells above covered glasses were fixed, permeabilized, and labeled with a specific rabbit anti-Tyr³⁹⁷ p-FAK primary antibody and with PE secondary antibody (red signals). 4′,6-diamidino-2-phenylindole (DAPI) was used to determine cellular nuclei (blue cell nuclei). Bar, 10 µm. (F) Bar graph representing the mean fluorescence of FC experiments showing expression levels of p-AKT and p-ERK1/2 proteins in Z138CT and Z138KD cultured overnight alone (−CC) or in cocultures with SNKT-GFP⁺ cells (+CC) and pretreated with PBS (Ø) or with the FAK inhibitor (+PF). Results are shown as mean fluorescence expression relative to Z138CT cultured overnight alone and pretreated with PBS (Z138CT −CC Ø). Bar plot represents the mean percentage ± standard deviation of 3 independent experiments. The significance of difference was determined by independent samples Student t test: *P < .05, **P < .01, ***P < .001.