Supplemental Figures 1-19 from ROS-Induced CXCR4 Signaling Regulates Mantle Cell Lymphoma (MCL) Cell Survival and Drug Resistance in the Bone Marrow Microenvironment via Autophagy

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<0.05; **, P<0.005. P values were calculated using Student's t-test. (B) AMD3100 treatment abolishes the protective effects of the stromal cells against chemotherapeutic drugs. A CXCR4 antagonist, AMD3100 (40 mM), was incubated with MCL cells for 24 hours, and cell viability was determined by CellTiter-Blue. The results represent the mean {plus minus} standard deviation of triplicates. *, P<0.05, **, P<0.005; P values were calculated using Student's t-test. Figure S9: Bortezomib treatment induces CXCR4 expression in MCL cells. Bortezomib-resistant Mino and REC1 cells (106, 6-well plate) were treated with different doses of bortezomib (0-100 nM for 24 hours). CXCR4 expression was analyzed by FACS. Figure S10: ROS generated after bortezomib treatment induces CXCR4 upregulation in MCL cells. Bortezomib-resistant Mino and REC1 cells (106, 6-well plate) were treated with bortezomib (30 nM for 24 hours) with or without NAC (100 mM, 1 hour). CXCR4 expression was evaluated using FACS. Figure S11: Ibrutinib 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 S12: HS27a stromal cells as well as HS27a media protect MCL cells from Ibrutinib 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. Ibrutinib was serially diluted (0-40 nM) as indicated. The results represent the mean {plus minus} standard deviation of triplicates. *, P<0.05; **, P<0.005. P values were calculated using Student's t-test. Figure S13: ROS weren't generated after Ibrutinib treatment. Jeko and SP53 cells (106, 6-well plate) were treated with Ibrutinib (0, 20, 40 mM for 24 hours) and ROS was measured using DCH-FDA (Sigma-Aldrich). ROS was measured at Ex/Em, 480/520 nm and then analyzed by flow cytometry. Figure S14: Ibrutinib treatment did not induce CXCR4 upregulation in MCL cells. Jeko and SP53 cells (106, 6-well plate) were treated with Ibrutinib (0, 20 and 40 mM) and CXCR4 expression was evaluated using FACS. Figure S15: A model diagram depicting observations made in the study. Ibrutinib treatment does not produce ROS as well as does not induce upregulation of CXCR4. Figure S16: Bortezomib (30 nM) was added to the MCL cells with or without lysosomal inhibitor chloroquine (50 mM), and autophagy induction was analyzed based on the LC3B-I to LC3B-II conversion as measured by immunoblots. Figure S17: Beclin1 was knocked down using lentivirus mediated shRNA in Jeko cells and Beclin1 levels were evaluated using immunoblots. GFP+ cells were selected followed by antibiotic selection to ensure knock down. Densitometer analysis showed a significant down-regualtion of beclin 1 in GFP+ Jeko cells compared to GFP+ scrambled shRNA infected Jeko cells. Figure S18: Jeko cells were cultured in low serum condition with AMD3100 to inhibit CXCR4 expression and autophagy expression was analyzed using immunoblots based on LC3-I to LC3-II conversion. Figure S19: Beclin 1 silencing does not change CXCR4 expression in MCL cells.