Has1 expression in myeloma bmMPCs after coculture with the plasma cell lines ARH77 and ANBL-6.
Multiple myeloma bone marrow bmMPCs were established as indicated in “Materials and methods.” When cultures reached 80% confluency, the medium was aspirated and 1 × 105 ANBL-6 and ARH77 cells were added to each well. Cocultures were incubated for 1, 6, and 12 hours, followed by treatment with 0.01% trypsin. RNA was isolated as described in “Materials and methods.” Coculture time course assays were done in quadruplicate, and RNA was pooled for each time point. Values indicated are the expression of Has1 relative to that observed in myeloma bmMPCs in the absence of coculture (100%). There were no significant differences in the total RNA values at the 1-, 6-, and 12-hour ARH77/ANBL-6 time points. Total RNA values at 1, 6, and 12 hours are shown for ARH77, and total RNA value at 1 hour is shown for ANBL-6. As such, and for consistency, all values were normalized to the 1-hour ARH77/ANBL-6 time point. SEM was less than 5% for all samples. (A) shows a representative histogram plot (representative of 3 experiments with 3 individual myeloma bmMPCs) for CD45-FITC–positive cells in ANBL-6:bmMPC cocultures after treatment with 0.01% trypsin at the (i) 1-hour and (ii) 12-hour time points. Staining above isotype-matched control mAb is represented by the marker in each histogram plot. BmMPCs do not express CD45 either as determined by flow cytometry (0% CD45-FITC–positive cells, data not shown) or as determined by immunohistochemistry.25 To assure that treatment with 0.01% trypsin did not result in the removal of CD45, U937 cells (CD45+) were treated with trypsin as indicated in “Materials and methods” and CD45-FITC expression was determined by flow cytometry. Prior to trypsin treatment, 70% U937 cells expressed CD45-FITC at a median channel of 147, whereas 75% of U937 cells expressed CD45 at a median channel of 151 after trypsin treatment.
