PCI-32765 affects gene expression in OC lineage cells activated with M-CSF/RANKL
| . | Donor 1 . | Donor 2 . | ||||||
|---|---|---|---|---|---|---|---|---|
| Fold increase by M-CSF and RANKL . | % inhibition by PCI-32765 (10nM) . | Fold increase by M-CSF and RANKL . | % inhibition by PCI-32765 (10nM) . | |||||
| Day 1 . | Day 7 . | Day 1 . | Day 7 . | Day 1 . | Day 7 . | Day 1 . | Day 7 . | |
| Btk | 6.44 ± 0.83 | 14.18 ± 1.36 | 0 | 23.14 | 7.50 ± 0.62 | 11.51 ± 0.92 | 0 | 0 |
| Syk | 3.16 ± 0.79 | 16.38 ± 0.78 | 0 | 29.30 | 3.68 ± 0.14 | 6.99 ± 0.86 | 0 | 0 |
| RANK | 8.71 ± 2.63 | 13.05 ± 0.50 | 0 | 0 | 6.25 ± 0.12 | 6.89 ± 0.18 | 0 | 0 |
| TRAF2 | 11.43 ± 1.35 | 15.61 ± 0.71 | 0 | 75.05 | 11.35 ± 0.15 | 3.72 ± 0.20 | 0 | 16.13 |
| MAPK8 | 4.73 ± 0.91 | 4.6 ± 0.14 | 0 | 39.08 | 4.75 ± 0.26 | 3.56 ± 1.23 | 0 | 0 |
| BAFF | 2.25 ± 0.6 | 14.56 ± 0.36 | 0 | 54.32 | 3.5 ± 0.10 | 8.35 ± 0.72 | 0 | 20.74 |
| LAT | 3.5 ± 0.74 | 24.74 ± 0.39 | 0 | 59.78 | 3.77 ± 0.56 | 8.39 ± 0.49 | 0 | 18.51 |
| NFATc1 | 16.18 ± 2.10 | 29.37 ± 1.34 | 0 | 27.00 | 23.66 ± 1.19 | 15.23 ± 0.52 | 29.14 | 0 |
| I-κB | 4.23 ± 0.43 | 6.16 ± 0.31 | 0 | 36.49 | 4.87 ± 0.14 | 3.64 ± 0.72 | 0 | 0 |
| APRIL | 1.62 ± 0.34 | 4.21 ± 0.04 | 0 | 0 | 2.52 ± 0.14 | 4.32 ± 0.62 | 0 | 0 |
| RANTES | 1.29 ± 0.10 | 7.44 ± 0.15 | 0 | 70.98 | 0.97 ± 0.01 | 2.61 ± 0.23 | 0 | 0 |
| NF-κB1 | 5.97 ± 0.99 | 8.41 ± 0.08 | 0 | 0 | 7.32 ± 0.75 | 6.03 ± 0.40 | 0 | 0 |
| IL-8 | 20.91 ± 4.29 | 13.65 ± 0.45 | 0 | 87.00 | 13.64 ± 0.81 | 2.02 ± 0.17 | 32.12 | 0 |
| MAPK14 | 2.08 ± 0.03 | 3.22 ± 0.26 | 0 | 0 | 2.49 ± 0.09 | 2.08 ± 0.31 | 0 | 0 |
| IL-6* | 1.08 ± 0.05* | 0.96 ± 0.04* | 0 | 0 | 1.19 ± 0.08* | 1.05 ± 0.05* | 0 | 0 |
| MIP1-β | 15.65 ± 2.74 | 5.37 ± 0.88 | 59.91 | 87.00 | 7.52 ± 0.62 | 4.48 ± 0.35 | 80.92 | 47.65 |
| MIP1-α | 12.10 ± 2.64 | 29.03 ± 0.77 | 75.25 | 84.04 | 10.35 ± 1.09 | 7.83 ± 1.41 | 76.61 | 54.02 |
| STAT3 | 4.25 ± 0.60 | 14.53 ± 1.83 | 0 | 30.17 | 4.86 ± 0.07 | 6.41 ± 0.35 | 0 | 0 |
| CHUK | 5.60 ± 0.48 | 5.43 ± 0.24 | 0 | 35.60 | 7.25 ± 0.17 | 4.31 ± 0.08 | 0 | 0 |
| CXCR4 | 0.79 ± 0.16 | 17.27 ± 0.02 | 0 | 57.20 | 0.80 ± 0.04 | 6.66 ± 1.43 | 0 | 13.95 |
| TGF-β1 | 4.82 ± 0.65 | 24 ± 0.67 | 0 | 52.22 | 6.41 ± 0.28 | 12.30 ± 0.45 | 0 | 13.15 |
| MAPK1 | 2.99 ± 0.87 | 12.43 ± 0.17 | 0 | 25.60 | 2.30 ± 0.06 | 8.02 ± 1.27 | 0 | 0 |
| BLNK | 2.52 ± 0.02 | 4.38 ± 0.02 | 0 | 38.81 | 3.37 ± 0.43 | 2.56 ± 0.13 | 0 | 0 |
| MYD88 | 3.12 ± 0.37 | 7.82 ± 1.41 | 0 | 50.87 | 4.13 ± 0.00 | 5.07 ± 0.16 | 0 | 13.15 |
| AKT1 | 3.28 ± 0.37 | 10.43 ± 0.74 | 0 | 28.47 | 3.78 ± 0.09 | 7.43 ± 0.29 | 0 | 0 |
| TRAF6 | 3.56 ± 0.65 | 6.06 ± 1.26 | 0 | 27.92 | 4.40 ± 0.55 | 3.89 ± 1.04 | 0 | 0 |
| PLC-β2 | 4.07 ± 0.24 | NA | 0 | NA | 5.55 ± 0.28 | NA | 0 | NA |
| PLC-γ2 | NA | 12.71 ± 0.75 | NA | 46.84 | NA | 3.77 ± 0.05 | NA | 0 |
| BLK* | 1.02 ± 0.01* | 1.00 ± 0.08* | ND | ND | 0.97 ± 0.05* | 1.10 ± 0.2* | ND | ND |
| PLC-γ1* | 1.31 ± 0.06* | 1.48 ± 0.17* | ND | ND | 1.49 ± 0.18* | 2.11 ± 0.63* | ND | ND |
| BMX* | 1.00 ± 0.06* | 0.81 ± 0.01* | ND | ND | 0.81 ± 0.01* | 1.10 ± 0.03* | ND | ND |
| RANKL* | 0.99 ± 0.04* | 0.94 ± 0.05* | ND | ND | 1.04 ± 0.09* | 0.93 ± 0.06* | ND | ND |
| . | Donor 1 . | Donor 2 . | ||||||
|---|---|---|---|---|---|---|---|---|
| Fold increase by M-CSF and RANKL . | % inhibition by PCI-32765 (10nM) . | Fold increase by M-CSF and RANKL . | % inhibition by PCI-32765 (10nM) . | |||||
| Day 1 . | Day 7 . | Day 1 . | Day 7 . | Day 1 . | Day 7 . | Day 1 . | Day 7 . | |
| Btk | 6.44 ± 0.83 | 14.18 ± 1.36 | 0 | 23.14 | 7.50 ± 0.62 | 11.51 ± 0.92 | 0 | 0 |
| Syk | 3.16 ± 0.79 | 16.38 ± 0.78 | 0 | 29.30 | 3.68 ± 0.14 | 6.99 ± 0.86 | 0 | 0 |
| RANK | 8.71 ± 2.63 | 13.05 ± 0.50 | 0 | 0 | 6.25 ± 0.12 | 6.89 ± 0.18 | 0 | 0 |
| TRAF2 | 11.43 ± 1.35 | 15.61 ± 0.71 | 0 | 75.05 | 11.35 ± 0.15 | 3.72 ± 0.20 | 0 | 16.13 |
| MAPK8 | 4.73 ± 0.91 | 4.6 ± 0.14 | 0 | 39.08 | 4.75 ± 0.26 | 3.56 ± 1.23 | 0 | 0 |
| BAFF | 2.25 ± 0.6 | 14.56 ± 0.36 | 0 | 54.32 | 3.5 ± 0.10 | 8.35 ± 0.72 | 0 | 20.74 |
| LAT | 3.5 ± 0.74 | 24.74 ± 0.39 | 0 | 59.78 | 3.77 ± 0.56 | 8.39 ± 0.49 | 0 | 18.51 |
| NFATc1 | 16.18 ± 2.10 | 29.37 ± 1.34 | 0 | 27.00 | 23.66 ± 1.19 | 15.23 ± 0.52 | 29.14 | 0 |
| I-κB | 4.23 ± 0.43 | 6.16 ± 0.31 | 0 | 36.49 | 4.87 ± 0.14 | 3.64 ± 0.72 | 0 | 0 |
| APRIL | 1.62 ± 0.34 | 4.21 ± 0.04 | 0 | 0 | 2.52 ± 0.14 | 4.32 ± 0.62 | 0 | 0 |
| RANTES | 1.29 ± 0.10 | 7.44 ± 0.15 | 0 | 70.98 | 0.97 ± 0.01 | 2.61 ± 0.23 | 0 | 0 |
| NF-κB1 | 5.97 ± 0.99 | 8.41 ± 0.08 | 0 | 0 | 7.32 ± 0.75 | 6.03 ± 0.40 | 0 | 0 |
| IL-8 | 20.91 ± 4.29 | 13.65 ± 0.45 | 0 | 87.00 | 13.64 ± 0.81 | 2.02 ± 0.17 | 32.12 | 0 |
| MAPK14 | 2.08 ± 0.03 | 3.22 ± 0.26 | 0 | 0 | 2.49 ± 0.09 | 2.08 ± 0.31 | 0 | 0 |
| IL-6* | 1.08 ± 0.05* | 0.96 ± 0.04* | 0 | 0 | 1.19 ± 0.08* | 1.05 ± 0.05* | 0 | 0 |
| MIP1-β | 15.65 ± 2.74 | 5.37 ± 0.88 | 59.91 | 87.00 | 7.52 ± 0.62 | 4.48 ± 0.35 | 80.92 | 47.65 |
| MIP1-α | 12.10 ± 2.64 | 29.03 ± 0.77 | 75.25 | 84.04 | 10.35 ± 1.09 | 7.83 ± 1.41 | 76.61 | 54.02 |
| STAT3 | 4.25 ± 0.60 | 14.53 ± 1.83 | 0 | 30.17 | 4.86 ± 0.07 | 6.41 ± 0.35 | 0 | 0 |
| CHUK | 5.60 ± 0.48 | 5.43 ± 0.24 | 0 | 35.60 | 7.25 ± 0.17 | 4.31 ± 0.08 | 0 | 0 |
| CXCR4 | 0.79 ± 0.16 | 17.27 ± 0.02 | 0 | 57.20 | 0.80 ± 0.04 | 6.66 ± 1.43 | 0 | 13.95 |
| TGF-β1 | 4.82 ± 0.65 | 24 ± 0.67 | 0 | 52.22 | 6.41 ± 0.28 | 12.30 ± 0.45 | 0 | 13.15 |
| MAPK1 | 2.99 ± 0.87 | 12.43 ± 0.17 | 0 | 25.60 | 2.30 ± 0.06 | 8.02 ± 1.27 | 0 | 0 |
| BLNK | 2.52 ± 0.02 | 4.38 ± 0.02 | 0 | 38.81 | 3.37 ± 0.43 | 2.56 ± 0.13 | 0 | 0 |
| MYD88 | 3.12 ± 0.37 | 7.82 ± 1.41 | 0 | 50.87 | 4.13 ± 0.00 | 5.07 ± 0.16 | 0 | 13.15 |
| AKT1 | 3.28 ± 0.37 | 10.43 ± 0.74 | 0 | 28.47 | 3.78 ± 0.09 | 7.43 ± 0.29 | 0 | 0 |
| TRAF6 | 3.56 ± 0.65 | 6.06 ± 1.26 | 0 | 27.92 | 4.40 ± 0.55 | 3.89 ± 1.04 | 0 | 0 |
| PLC-β2 | 4.07 ± 0.24 | NA | 0 | NA | 5.55 ± 0.28 | NA | 0 | NA |
| PLC-γ2 | NA | 12.71 ± 0.75 | NA | 46.84 | NA | 3.77 ± 0.05 | NA | 0 |
| BLK* | 1.02 ± 0.01* | 1.00 ± 0.08* | ND | ND | 0.97 ± 0.05* | 1.10 ± 0.2* | ND | ND |
| PLC-γ1* | 1.31 ± 0.06* | 1.48 ± 0.17* | ND | ND | 1.49 ± 0.18* | 2.11 ± 0.63* | ND | ND |
| BMX* | 1.00 ± 0.06* | 0.81 ± 0.01* | ND | ND | 0.81 ± 0.01* | 1.10 ± 0.03* | ND | ND |
| RANKL* | 0.99 ± 0.04* | 0.94 ± 0.05* | ND | ND | 1.04 ± 0.09* | 0.93 ± 0.06* | ND | ND |
CD14+ OC precursor cells from 2 normal donors were plated (6 × 104 cells per well) in 96-well plates and stimulated with M-CSF/RANKL for 1 and 7 days in the presence or absence of PCI-32765 (10nM). Cells were harvested and subject to QuantiGene Expression Assay to determine gene expression at each condition. Expression levels were normalized with the internal control HPRT. % inhibition on gene expression by PCI-32765 was determined as follows: % inhibition = 100 × [1 − (expression with PCI-32765 and cytokine stimulation)/(expression with cytokine stimulation)].
0 indicates no inhibition; NA, not available; and ND, not determined due to insignificant expression level.
Gene expression is neglected because of low level.