Fig. 4.

Stable transfection of SCC-61 parental cell line by Stat1α and Stat1β cDNAs leads to increased radioprotection of the transfected clones. (A) Protein expression of Stat1 in stably transfected clones. pAttBStat1α, pAttBStat1β, or pAttB were cotransfected into SCC-61 with pCMV-INT, kindly provided by Michele Calos. Cells were selected in the Hyg-containing media, and selected monoclones were further propagated for experiments (see Materials and Methods). Several clones, transfected by pAttBStat1β, pAttBStat1α, or pAttB (empty vector), were checked by PCR of genomic DNA for appropriate integration and checked by Western analysis for expression of α and β isoforms of Stat1. b1, b2, b3, and b11 correspond to the clones stably transfected by pAttBStat1β. a1, a2, a5, a12, and a16 correspond to the clones stably transfected by pAttBStat1α. ev2, ev5, and ev7 correspond to the clones, stably transfected by pAttB and related as “empty vector.” For the further experiments we choose b1, b2, and a16 clones as Stat1 stable transfectants and ev5 clone as the “empty vector” control. (B) Stably transfected α and β clones protect parental cell line from ionizing radiation. SCC-61, nu61, and b1, b2, a16, and ev5 clones were plated in the 96-well plates and either irradiated at 3 or 10 Gy (see Materials and Methods) or left untreated. All experiments were made in triplicates. Viability of irradiated and untreated cells was assessed by the MTS assay 72 h after irradiation (see Materials and Methods). Fraction of surviving cells was calculated as percent of viable cells after irradiation to unirradiated control for the each type of the cells. For the stable transfectants of the SCC-61, significance of survival was calculated relative to the ev5 clone, marked by asterisks. P values were calculated as the two-tailed, unpaired t test and are indicated in B above each corresponding bar. Error bars are standard deviations of triplicate experiments. (C) Transient transfection of the parental cell line SCC-61 by plasmids, carrying Stat1β and Stat1α leads to the increased radioprotection. SCC-61 cells were transfected with the pAttBStat1α, pAttBStat1β or pAttB plasmids (see above and Materials and Methods). As the mock we used SCC-61, treated in the same way as transfected cells but without exogenous plasmids. Twenty-four hours after transfection, cells were irradiated at 3 Gy, and 24 h after irradiation viable cells were scored by the Trypan Blue assay (see Materials and Methods). Surviving fraction was calculated as the percent of viable cells after irradiation to unirradiated control for the each type of the cells. Significance was calculated relative to the ev5-transfected cells (marked by asterisk), using two-tailed unpaired t test. P values are indicated above the appropriate columns. Error bars are standard deviations of the triplicate experiments.