Fig. 2
Tau silencing reduces the survival of CDA-deficient cells. a MAPT mRNA levels determined by RT-qPCR in BS-Ctrl(BLM) and BS-BLM cells. b, c BLM and Tau levels determined by western blotting in BS-Ctrl(BLM) and BS-BLM cells using BLM and either Tau-5 (b) or Tau-1 (c) antibodies. d, e Tau protein quantification for Tau-5 (d) and Tau-1 (e) immunoreactivity. The β-actin signal was used as the control. The results are normalized against those for BS-BLM, which were set to 1. f–k Cells were first transfected with either non-targeting or Tau siRNA. After 24 h, cells were again transfected with the indicated siRNAs. Two days after the second round of transfection, the cells were collected for western blotting f, i, or were plated, in a serial dilution series, in 12-well plates. Ten to 12 days later, colonies were fixed and stained with crystal violet. Non-targeting siRNA was used as a control, with values set to 1 h, k. g, j Tau protein quantification after Tau-targeting siRNA depletion. l MAPT mRNA and m Tau protein levels determined in HeLa-Ctrl(Tau) and HeLa-shTau cells. n, o HeLa-Ctrl(Tau) and HeLa-shTau cells were first transfected with the indicated siRNAs. After 24 h, cells were again transfected with the same siRNAs. Two days after the second round of transfection, cells were collected for western blotting (n), or were plated, in a serial dilution series, in 12-well plates. Seven days later, colonies were fixed and stained with crystal violet. Non-targeting siRNA was used as a control, with the values obtained set to 1 (o). For qPCR, the mean value for 3 independent experiments is represented as a fold-change. B2M, β-actin, and TBP were used as housekeeping genes for qPCR normalization. For western blots, β-actin was used as a loading control. Each data bar is the mean of at least three independent experiments performed in triplicate. Error bars represent ± SEM. The significance of differences was assessed in two-tailed paired Student’s t-tests. ***P < 0.0005, **P < 0.005, *P < 0.05, NS not significant
