TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression -- Ayala et al. 105 (10): 3785 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Ayala et al. 10.1073/pnas.0800546105.

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SI Figure 5

Fig. 5. Cdk6 up-regulation and nuclear membrane disruption are observed upon TDP-43 depletion with siRNA oligonucleotide T1. (A) Immunoblotting to detect TDP-43 and Cdk6 in cell extracts from T1 siRNA-depleted and control-treated HeLa. Tubulin was used as loading control. (B) Indirect immunofluorescence detecting lamin A/C and TDP-43 in RNAi-treated HeLa cells using control, T2, or T1 siRNA oligonucleotides.

SI Figure 6

Fig. 6. siRNA-resistant TDP-43 partially offsets CDK6 up-regulation resulting from endogenous TDP-43 silencing. HeLa cells were transfected with control siRNA or TDP-43-specific siRNA in addition to control pFLAG vector or FLAG-tagged TDP-43 mutated to resist siRNA depletion (TDP-43^siR). The levels of endogenous TDP-43, Cdk6, and FLAG-TDP-43^R were determined by Western blotting and quantified by Image J software. Tubulin was used as loading control. Shown is a representative result of three independent experiments. Cdk6 protein levels after depletion of TDP-43 were 35% to 45% lower in the presence of TDP-43^siR compared to control-treated cells. The lack of complete recovery of normal CDK6 levels may be explained by the lower transfection efficiency of large plasmids (TDP-43^siR) compared to short oligonucleotides (siRNA^TDP-43).

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Fig. 7. pRb phosphorylation does not change upon TDP-43 silencing in chicken cells, where CDK6 levels are unresponsive to TDP-43 loss. DF-1 chicken embryo fibroblasts were depleted of TDP-43 by RNAi. Immunoblotting was performed on lysates from siRNA and control-treated cells to detect pRb and pRb2/p130 phosphorylation. Tubulin was used as loading control.

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Fig. 8. DNA damage and activation of apoptosis detected upon TDP-43 silencing. (A) Confocal images of TDP-43-depleted and control-treated nuclei immunolabeled with phosphorylated histone H2AX (gH2AX) in HeLa cells. The dotted line indicates the location of the nuclei. (Scale bar, 10 mM.) (B) Immunoblotting of PARP-1 comparing U2OS cell extracts from control (cont.), staurosporine (STS)-treated cells, and cells transfected with control or specific siRNA. Cells were treated with 1 mM staurosporine for 5 h as control for PARP-1 cleavage. Tubulin was used as loading control.

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Fig. 9. Depletion of TDP-43 in p53- cells, MG-63, leads to dysmorphic nuclei and apoptosis. Osteosarcoma cells defective in p53, MG-63, were depleted of TDP-43 by RNAi treatment. Nuclei were visualized by lamin A/C in the absence of TDP-43 (siRNA^TDP-43) and compared to control-treated cells (siRNA^control).