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. Author manuscript; available in PMC: 2019 Dec 6.
Published in final edited form as: Mol Cell. 2018 Nov 8;72(5):823–835.e5. doi: 10.1016/j.molcel.2018.09.019

Figure 1: Silencing USP46 or E6 decreases Cdt2 in HPV E6 transformed cells.

Figure 1:

(A) U2OS and HeLa cells were transfected with GL2 (control siRNA) or siRNAs against E6 or E6AP and harvested after 60 hrs. The cell lysates were immunoblotted as indicated. (B) Different siRNAs towards 18E6 (lane 2, 4 and 6) were transfected into HeLa cells and immunoblotted. (C) HeLa cells were transfected with GL2 (control) or 18E6 siRNAs and immunoblotted. Lanes 3 and 4: Cells exposed to 10 μM MG132 for 8 hrs before harvest. (D) Cdt2 and p21 mRNA levels measured by RT-qPCR and normalized to actin mRNA after transfection of indicated siRNA into HeLa cells. Mean ± standard deviation of three replicates (E) HeLa cells were transfected with control siRNA (GL2) or with siRNAs against 18E6 or p53 for 60 hrs and lysates probed with indicated antibodies. (F) Indicated cell lines were transfected with GL2 (control) or USP46 siRNA, harvested after 60 hrs and immunoblotted. Actin was used as loading control. (G) Indicated cell lines were transfected with GL2 (control) or E6 siRNAs, harvested after 60 hrs and immunoblotted. Actin was used as loading control. (H) Indicated cell lines were transfected with GL2 (control) or USP46 siRNA, harvested after 60 hrs and immunoblotted. Actin was used as loading control. (I) Lysates of primary human keratinocytes (PHK) ± HPV18 or U2OS ± plasmid expressing HPV18 E6 were immunoblotted as indicated. (J) 293T cells transfected with indicated siRNAs were transfected with empty vector (V) or plasmid expressing HPV16-E6 and 72hrs after siRNA transfection, cell lysates were immunoblotted for indicated antibodies. (K) U2OS cells, transfected with empty vector or HPV-16 E6 expressing plasmid were enriched in S phase by serum starvation for 48 hours and then released into serum for 8 hr. The cell lysates were then probed with indicated antibodies. Indicated siRNAs were transfected 18 hours before collection.