Skip to main content
. 2015 Feb 17;6(1):e02068-14. doi: 10.1128/mBio.02068-14

FIG 5 .

FIG 5 

HSP90, GRP78, and E6^E7 are required to maintain a steady-state level of E6 and E7 in HPV16-positive CaSki cells. (A to C) A functional HSP90 is required for the stability of HPV16 E6 and E7 and proliferation of cervical cancer cells. HPV16-positive CaSki cells treated with 5 µM 17-AAG or DMSO for 48 h were examined by Western blotting (A), RT-PCR (B), and a cell proliferation assay (C). p53 was used to indicate E6. GAPDH served as a loading control in RT-PCR. (D and E) Knockdown of GRP78 expression in CaSki cells destabilizes E6 and E7 and inhibits cell growth. CaSki cells treated twice with 40 nM nontargeting siRNA (si-NS) or GRP78 siRNA (si-GRP78) for 96 h were examined by Western blotting (D) and a cell proliferation assay (E). (F to I) Knockdown of E6^E7 expression in CaSki cells destabilizes viral E6 and E7 and prevents cell growth. An E6^E7-specific siRNA (si-E6^E7) for the nt 226-to-nt 742 splice junction (F) was designed to silence E6^E7 expression without affecting other E6 splice isoform RNAs, as shown by RT-PCR (G). CaSki cells transfected twice with 40 nM si-NS or si-E6^E7 at 48-h intervals for 96 h were examined for E6 (p53) and E7 expression (H) and cell proliferation (I). See the details in panels A to C. (J and K) Overexpression of E6^E7 in CaSki cells increases E7 stability (J) and promotes cell proliferation (K). CaSki cells were transfected twice (for Western blotting at day 5) or three times (for cell proliferation at day 7) with a FLAG-E6^E7 or an empty vector at 24-h intervals. (L and M) Overexpression of E6^E7 has no effect on C33A, an HPV-negative cervical cancer cell line containing mutations in both p53 and pRB. C33A cells transfected with a FLAG-E6^E7 or an empty vector as described for panels J and K served as a cell line control to CaSki cells, and results were analyzed by Western blotting (L) and a cell proliferation assay (M) at days 5 and 7, respectively. *, P < 0.05; **, P < 0.01. Nonsignificance (N/S), P ≥ 0.05 by Student’s t test (C and E, I and K, and M). (A, D, H, J, and L) β-Actin served as a sample loading control.