Subcellular fractions Cytoplasmic and nuclear extracts have been prepared accord ing to your instructions contained while in the NE PER Nuclear and Cytoplasmic Extraction Reagent Kit. siRNA transfection Cells had been transfected with 50 nM nontargeting siRNA or unique siRNA using Lipofectamine 2000 transfection reagent in accordance to the protocol of the manufacturer. Twenty four hrs just after transfection the media have been transformed. Cells have been employed for experiments four days soon after transfection. For knockdown of YB 1, cells had been trans fected with YB 1 siRNAI/II and for knockdown of K Ras, a K RAS precise pool of siRNA was made use of. Sequencing of KRAS Complete RNA was isolated from frozen cell pellets working with the RNeasy mini kit and reverse transcribed with the Reverse iT 1st Strand Synthesis Kit making use of anchored oligo primers. Exons 1 to three of K RAS have been ampli fied from the cDNA working with ReddyMix PCR Master Combine with specific primers, and both strands have been sequenced by a business subcon tractor.
K RASV12 overexpression Subconfluent K RASwt cells had been trypsinized, selleck chemicals and 2 ? 106 cells have been transiently trans fected with 5 ug of p EGFP C1 manage vector or p EGFP/K RASV12 by way of electroporation. Following 24 hrs, the efficiency of transfection was tested by fluor escent microscopy of green fluorescent protein, and thereafter the media have been changed. Immediately after an addi tional 24 hrs, cells have been used for experiments. g H2AX foci formation assay The g H2AX foci formation assay was applied to evaluate residual DNA DSB as described previously. Briefly, the cells have been cultured on coverglass slides and trans fected with inhibitor I-BET151 50 nM nontargeting siRNA or specific siRNA against YB 1 and K RAS. Right after 24 hrs, the medium was exchanged with fresh medium. Forty eight hours later the cells were exposed to single doses of irradiation of 2, four, and six Gy and incubated at 37 C for an additional 24 hours.
Thereafter the slides have been stained with phospho H2AX as described pre viously. The g H2AX foci were counted and graphed. Clonogenic assay Clonogenic cell survival following radiation exposure was analyzed by way of colony formation assay. Cells had been preplated in six very well plates and 24 hrs later on have been mock irradiated or irradiated with single doses of one, one. five, 2, three or four Gy. Irradiation was performed at 37 C applying a Gulmay RS225 X ray machine which has a dose fee of 1. seven Gy/minute as well as the exposure factors of 150 kVp, 15 mA and 0. three mm Al extra filtering. To investigate the effect of YB one expression on postirradiation survival, cells were transfected with nontargeting siRNA or YB 1 unique siRNA. 3 days immediately after transfection cells have been preplated in six effectively plates, and 24 hrs later the cells had been mock irradiated or irradiated with single doses of 1, one.