1. Supplementary 3 PTB PTB Actin Actin 0.014 GM1953 control 0.03
GM1953 siPTB
0.012
0.025
GM1311 siPTB
0.01
0.02
0.008
CPM/cell number
CPM/cell number
0.015
0.006
0.01
0.004
0.002
0.005 6 12 18 24 6 12 18 24
Time (hrs)
Time (hours)
PTB levels were reduced by siRNA and cells were seeded into 12 well plates at 100,000 cells/well. After 24 hours, 5µCi/ml of [3H]thymidine was added to each well with fresh media, and then cells were incubated and harvested at 6 hour intervals. The cells were then washed twice with phosphate buffered saline before the addition of 10 % (w/v) trichloroacetic acid. Cells were collected and precipitated on ice for 30 min before being filtered through nitrocellulose with extensive washing. Filters were dried, and scintillation readings taken on a Molecular Dynamics scintillation counter. Cell proliferation is expressed as [3H]thymidine incorporation (CPM) relative to cell number. The data show that in GM1311 the MM derived cell line when the expression of PTB-1 is reduced proliferation is inhibited more than the control B-cell line GM1953.
Supplementary 3