Knockdown of c-Jun and JunB reduces tumor size in a xenograft model of DLBCL and lymphoma cell invasiveness. (A) Xenograft model of DLBCL. Nude mice (n = 5) were inoculated with OCI-Ly3 cells (5 × 106) stably expressing shGFP (right site) and shJunB/c-Jun (left site). Tumors were measured weekly with calipers, and tumor weight was determined at week 7. (B) Enlarged lymph nodes in nude mice inoculated subcutaneously with OCI-Ly3 cells expressing the indicated shRNAs. Lymphoma cells were identified by immunostaining (IHC) with human CD20 antibody (Ab; brown). Pictures were taken with the Olympus IX70 inverted fluorescence microscope (×40). (C) Subcutaneous tumors in nude mice. Control cells or cells with silenced JunB/c-Jun were injected into experimental mice (n = 8 per group), and tumor growth was monitored as described in (A). (D) Bones (femurs and tibias) were collected from experimental mice, and bone marrow infiltration by CD20+ cells was quantified by using the Automated Cellular Imaging System (ACIS III, Dako). Results are presented as relative intensity level in mice that developed tumors of comparable size (labeled as #1, #2, and #3). (E) In vitro invasion assay was performed by using transwell inserts coated with Matrigel. Results are presented as mean + SD of triplicate cultures. (F) Lymphoma cell lines were labeled with CMFDA (CellTracker Green, Molecular Probes) for 30 minutes and injected into the recipient mice (106 cells per mouse). Bone marrow infiltration was determined by flow cytometry 16 hours later. Results are presented as mean + SD of duplicates. (G) Lymphoma cell adhesion to bone marrow stroma. Freshly isolated bone marrow stromal cells were seeded in 6-well plates. OCI-Ly3 cells stably expressing shGFP (control) or shJunB plus c-Jun were labeled with 5-chloromethylfluorescein diacetate (CellTracker Green) for 30 minutes and cocultured with a monolayer of stromal cells for 3 hours. The pictures were taken after 3 washes with warm PBS.
