Figure 1.
E7 expressed from episomes localises in the cytoplasm in confluent cells.
(a) Immunofluorescence of HPV16E7 protein in sub-confluent and confluent NIKS+HPV16 cells. NIKS cells (with no HPV16) were analysed in parallel as a negative control. (b) Confocal microscopy of HPV16E7 protein in sub-confluent and confluent NIKS+HPV16 cells.
Figure 2.
E7 expressed alone localises in the cytoplasm in confluent cells.
Immunofluorescence of HPV16E7 protein in sub-confluent and confluent NIKS+E7 cells (only expressing E7). NIKS cells (vector control) were analysed in parallel as a negative control.
Figure 3.
E7 in cell lines derived from a pre-cancerous lesion and naturally occurring cancers localises in the cytoplasm in confluent cells.
Immunofluorescene of HPV16E7 protein in sub-confluent and confluent (a) W12, (b) CaSki and (c) SiHa cells.
Figure 4.
Levels of E7 increase in confluent cells.
Western blotting of HPV16E7 protein in sub-confluent and confluent NIKS and NIKS+HPV16 using (a) whole cell lysates and (b) nuclear and cytoplasmic fractions. HSP70 used as a loading control and HDAC2 and GAPDH as fractionation controls.
Figure 5.
Blocking nuclear export inhibits cytoplasmic localisation of E7 in confluent cells.
(a) Immunofluorescence of cyclin B1 used as a positive control for nuclear export inhibition in confluent NIKS+HPV16 cells treated with leptomycin B. (b) Immunofluorescence of HPV16E7 protein in confluent NIKS+HPV16 cells treated with leptomycin B to block nuclear export. NIKS control cells (with no HPV16) treated with leptomycin B were analysed in parallel.
Figure 6.
Localisation of E7 is not linked to cellular proliferation.
Sub-confluent and confluent NIKS and NIKS+HPV16 cells were treated with EdU (BrdU alternative) to label cells undergoing DNA synthesis. The cells were stained for EdU and the percentage of labelled cells was calculated.
Figure 7.
Cytoplasmic localisation of E7 is not linked to the cell cycle.
Immunofluorescence of HPV16E7 protein in sub-confluent NIKS+E7 treated with different compounds to arrest the cell cycle at different stages. (a) Confluent control (b) untreated sub-confluent control and sub-confluent (c) mimosine (G1 block), (d) thymidine (S-phase block), (e) etoposide (G2 block) and (f) nocodazole (mitosis block). Cell cycle blocks were confirmed in parallel by propidium iodide staining analysed by flow cytometry.
Figure 8.
Effect of E7 on pRb at sub-confluence and confluence.
Western blot analysis of pRb in sub-confluent and confluent NIKS and NIKS+E7 cells. HSP70 is shown as a loading control. Graphs show integrated density measured by ImageJ and normalised to HSP70 for the blots shown. Two replicates were analysed for the reduction of pRb and the error bars show the range of the data (i.e. the lowest and highest values).
Figure 9.
Effects of E7 on SRC-1 and p130 at sub-confluence and confluence.
Western blot analysis of (a) SRC-1 and (b) p130 in sub-confluent and confluent NIKS and NIKS+HPV16 cells. HSP70 is shown as a loading control. Graphs show integrated density measured by ImageJ and normalised to HSP70 for the blots shown. Three replicates were analysed for the reduction of SRC-1 and p130 and the error bars show standard deviation. On average the drop in levels of SRC-1 was 4.37 fold more and p130 was 7.40 fold more in confluent cells compared to sub-confluent cells.