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Figure S1: FACS analyses of CXCR4 silenced in Jeko, SP53 and Z138 MCL cells. GFP-positive cells were sorted after infection and CXCR4 expression was evaluated using FACS analyses. 94-74% reduction was noted in all knock down cells. Figure S2: Migration of MCL cells to SDF-1 was effectively decreased after CXCR4 silencing. GFP-positive controls or CXCR4 silenced MCL cells (105) were placed in upper chambers with different concentrations of SDF-1 in lower chambers and the cells were counted in the lower chamber after 24 hours. Figure S3: CXCR4 silenced cells showed reduction of migration toward the medium harvested from HS27a culture. Medium from HS27a was harvested after 3 days and 600 mL was added to transwell. GFP-positive controls or CXCR4 silenced MCL cells (Jeko, SP53, Z138 and REC1, 105) were placed in upper chambers with HS27a medium in lower chambers and cells were counted in the lower chambers after 24 hours. Figure S4: SDF-1 protects the MCL cells from starvation induced cell death but neutralized antibodies were reversed the effect of protection. MCL cells (1x106) were cultured in low serum for three days and SDF-1 (10ng/ml) or neutralizing SDF-1 antibodies were added to the culture. Cell death were evaluated based on 7-AAD/Annexin V staining using FACS. Figure S5: Colonies from PHA-LCM medium was harvested and light chain restriction was analyzed using fix and perm cell permeabilization kit (Invitrogen). Colonies harvested from PHA-LCM medium were lambda restricted similar to the SP53 parent cells. Figure S6: CXCR4 silencing decreases the motility of MCL cells. We quantitatively measured the distances the CXCR4-silenced or control cells travelled over 2 days using a time-lapse microscopy. Distances (mm) traveled by CXCR4-silenced SP53 and REC1 cells, which were an average of results from 15 different wells, were significantly decreased compared to the control cells. Figure S7: Bortezomib IC50 was calculated using different MCL cells. IC50 value (the concentration of a drug that is required for 50% inhibition in vitro) was used to indicate the quantitative measure of the different cell killing effect of drugs. The Hill-Slope logistic model is used to calculate IC50 using MS Excel. Figure S8A&B: (A) HS27a stromal cells as well as HS27a media protect MCL cells from chemotherapeutic drug cytotoxicity. CD19+ cells were isolated from four different patient cells (105) and were cultured with HS27a stromal cells or 1 ml of 1:1 diluted HS27a medium. Cell viability was determined by the CellTiter-Blue® fluorometric assay (Promega) and was indicated as a ratio compared to cell viability without treatment. Bortezomib was serially diluted (0-40 nM) as indicated. The results represent the mean {plus minus} standard deviation of triplicates. *, P |
