Fig 2: The overexpression of C65G tRNA[Ser]Sec was unable to restore selenoprotein expression in HAP1 C65G cells. WT and C65G HAP1 cells were grown in the presence or absence of 100 nM selenium prior to the transfection of a plasmid expressing either the WT or C65G tRNA[Ser]Sec. A control (Ctl) condition was used as a reference where an empty vector was used instead. GFP was systematically co-transfected to normalize the transfection efficiency. Two days post-transfection, the cells were harvested and split in half for either total RNA or cellular protein extraction. (A) A 15 μg aliquot of total RNAs was employed for the northern blot using tRNA[Ser]Sec (green) and tRNASer (red) complementary DNA probes for hybridization as described earlier. The levels of tRNA[Ser]Sec were normalized over those of tRNASer for three independent northern blots and expressed relative to the first lane. For clarity, only the fold changes between Ctl with WT and Ctl with C65G transfections are indicated. Error bars represent the SD. Mean values were tested for statistical significance by an unpaired Student's t-test (***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05). (B) A 40 μg aliquot of protein extracts was used for the western blots to evaluate the expression levels of SELENOS, SELENOH, GPX4, SELENOO, GPX1, TXNRD1, GFP and actin. The quantifications of three independent western blots were normalized by the signal for actin and expressed relative to those of lane 4, set as 1. The relative selenoprotein levels were measured and mean values were tested for statistical significance by an unpaired Student's t-test (***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, not significant).