![Figure 1. In vitro biologic activity of hIFN-α2b. (A) Activation of a classic IFN-inducible pathway. A multiplex RT-PCR analysis with primers specific for β-actin and 2′-5′oligoadenylate synthetase (2′-5′ OAS) performed using RNA extracted from hIFN-α2b-LV–transduced CRO-AP/3 cells at different MOI and from mock-transduced and EGFP-LV–transduced (MOI 25) cells 4 and 10 days after transduction showed the activation of 2′-5′ OAS expression only in hIFN-α2b-LV–transduced cells, indicating the specific activation of an IFN-inducible pathway. Lanes: M, 1-kb DNA ladder marker (Invitrogen); 1, water control; 2,7, RNA extracted from mock-transduced CRO-AP/3 cells 4 and 10 days after transduction; 3, RNA from EGFP-LV–transduced CRO-AP/3 cells (MOI 25) extracted 4 days after transduction; 4 to 6, RNA from CRO-AP/3 cells 4 days after transduction with the hIFN-α2b-LV using 3 MOIs (2, 10, and 25, respectively); 8 to 10, RNA from CRO-AP/3 cells 10 days after transduction with the hIFN-α2b-LV using 3 MOI. (B) Impaired proliferation of hIFN-α2b-LV–transduced CRO-AP/3 cells. [3H]-Thymidine incorporation was measured 10 days after transduction for 4 days, and a MOI-dependent reduction in cell proliferation was observed. Data are reported as ratio between the mean of triplicates of LV-transduced cells and the mean of triplicates of control EGFP-LV–transduced cells for each analyzed time point, and the SD of the ratio was calculated according to the theory of error propagation [σa/b = a/b√(σa/a)2 + (σb/b)2]. (C) Apoptosis induction in hIFN-α2b-LV–transduced CRO-AP/3 cells. Flow cytometric analyses after staining with annexin V/PI of hIFN-α2b-LV–transduced CRO-AP/3 cells showed an increase in apoptosis compared with control cells. A representative experiment is shown here, performed 18 days after transduction; density plot histograms of flow cytometric analysis show the percentage of total apoptosis (all annexin V–positive cells, reported in the circle) of the negative control (mock-transduced CRO-AP/3 cells) and hIFN-α2b-LV–transduced CRO-AP/3 cells.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/19/10.1182_blood-2008-09-180307/7/zh80170934190001.jpeg?Expires=1735587779&Signature=JqRsncXxkuoHsxsgzFvANM5UKtBk5oD178dMI5gTzMMb4UzvJSpwUi1S4xMbNs~-EObUGHdK99LGJnhSX1z3-NLbBAhxP2wV87oD~l1QhVnbiILXEIX7VA7QSpSvgp9fb6Yur96FRikfjcaC6rzQBU1ZyOrm6m0UpoKPG0xRsgB5aVLNwROKLsD7T4KSKNOTgAoMfhArDUB6sztxRmZWGspp3Xr-uE7fvZlyOGuIhwGqVIE-Ifmc4Ac4WEHOZ4oHSPkKA~J0J~HmY3Q9cyXFzbnFd9pT1fPfUuk7TFNv7RfybY~~kG8uYIWy~VujaxWPqm-gC8z9Y7su~3FIT1IVuQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
In vitro biologic activity of hIFN-α2b. (A) Activation of a classic IFN-inducible pathway. A multiplex RT-PCR analysis with primers specific for β-actin and 2′-5′oligoadenylate synthetase (2′-5′ OAS) performed using RNA extracted from hIFN-α2b-LV–transduced CRO-AP/3 cells at different MOI and from mock-transduced and EGFP-LV–transduced (MOI 25) cells 4 and 10 days after transduction showed the activation of 2′-5′ OAS expression only in hIFN-α2b-LV–transduced cells, indicating the specific activation of an IFN-inducible pathway. Lanes: M, 1-kb DNA ladder marker (Invitrogen); 1, water control; 2,7, RNA extracted from mock-transduced CRO-AP/3 cells 4 and 10 days after transduction; 3, RNA from EGFP-LV–transduced CRO-AP/3 cells (MOI 25) extracted 4 days after transduction; 4 to 6, RNA from CRO-AP/3 cells 4 days after transduction with the hIFN-α2b-LV using 3 MOIs (2, 10, and 25, respectively); 8 to 10, RNA from CRO-AP/3 cells 10 days after transduction with the hIFN-α2b-LV using 3 MOI. (B) Impaired proliferation of hIFN-α2b-LV–transduced CRO-AP/3 cells. [3H]-Thymidine incorporation was measured 10 days after transduction for 4 days, and a MOI-dependent reduction in cell proliferation was observed. Data are reported as ratio between the mean of triplicates of LV-transduced cells and the mean of triplicates of control EGFP-LV–transduced cells for each analyzed time point, and the SD of the ratio was calculated according to the theory of error propagation [σa/b = a/b√(σa/a)2 + (σb/b)2]. (C) Apoptosis induction in hIFN-α2b-LV–transduced CRO-AP/3 cells. Flow cytometric analyses after staining with annexin V/PI of hIFN-α2b-LV–transduced CRO-AP/3 cells showed an increase in apoptosis compared with control cells. A representative experiment is shown here, performed 18 days after transduction; density plot histograms of flow cytometric analysis show the percentage of total apoptosis (all annexin V–positive cells, reported in the circle) of the negative control (mock-transduced CRO-AP/3 cells) and hIFN-α2b-LV–transduced CRO-AP/3 cells.
