PMC

Venn diagram and Volcano plots representing the summary of microarray results. Figure A represents venn diagram, where each circle shows the main effects of Avadis analysis: A16 Vs NA16, A16 Vs Colony and A16 Vs Tumour. Numbers inside each compartment represents the number of transcripts that are significant for that effect. The intersections of the sets represent genes with P < 0.05 and Fold change 2 for each of the effects involved in the intersection. Note the expansion of transcripts that are significant and high from A16 Vs NA16 to A16 Vs Tumour. B, C and D represent Volcano plot comparison of gene expression between A16 Vs NA16, A16 Vs Colony and A16 Vs Tumour. The x axis indicates the differential expression profiles, plotting the fold-induction ratios in a log scale. The y axis indicates the statistical significance of the difference in expression (P value from a t test). Yellow color represents genes with P < 0.05 and Fold change 2. Squares within circles show potentially interesting genes from a biological standpoint. The increase in the fold change was observed from A16 to tumor cells and the genes that were obtained within the significant range were analysed through affymetrix database for different process based on functional annotations as shown in the Additional File 5 Figure S4.

Model of G9/A16 in MV membrane binding to A56/K2 in plasma membrane. G9 and A16 are anchored in MV membrane in association with the EFC. A56/K2 is anchored in plasma membrane through the transmembrane domain of A56. The interaction of A56/K2 with A16 and G9 is postulated to prevent fusion of the MV particle with the plasma membrane.

A26 interacts individually with A16 and G9 but not with G3 and L5 proteins in vitro. (A) GST-A26 pulled down A16 and G9 individually. HEK293T cells were transfected with plasmids expressing HA-tagged A16, Myc-tagged G9, or both and harvested 24 h later. The lysates were subjected to GST pulldown analyses using 30 μg of recombinant GST and GST-A26 proteins as described in Materials and Methods and analyzed by immunoblotting with anti-HA (A16) (1:1,000) and anti-Myc (G9) (1:1,000) antibodies. (B) GST-A26 did not pull down G3 and L5. HEK293T cells were transfected with plasmids expressing vaccinia G3 and L5 proteins and harvested 24 h later, and the lysates were subjected to GST pulldown analyses as described above using anti-G3 (1:1,000) and anti-L5 (1:1,000) antibodies. Input represents 1% of total cell lysates.

Permanganate reactivities of probes with TT insertions. (A) The A16 ori probe was modified by insertion of pairs of T's at different positions in the A16 sequence, as shown in panel C. These probes were tested for permanganate reactivity in the presence of 800 fmol of E1 as described in the legend to Fig. 1. For lanes 1, 3, 5, 7, 9, and 11, no ATP was added. For lanes 2, 4, 6, 8, 10, and 12, 5 mM ATP was added. (B) The bracketed part of the gel in panel A was scanned, and the scans for lanes 2, 4, 6, 8, 10, and 12 were aligned. (C) Summary of the permanganate reactivities generated with the TT-substituted A16 probes. The positions of the substitutions are boxed and shaded. Black bars indicate the positions and levels of permanganate reactivity observed on the A16 probe. The gray bars correspond to the pattern obtained after subtraction of the A16 pattern from each lane. (D) Bottom-strand labeled probes for the A16 template and the TT insertions T12/13, T14/15, T16/17, and T20/21 in the A16 context were tested for permanganate reactivity in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of 800 fmol of E1 in the presence of ADP. (E) Summary of the permanganate reactivities generated by E1 on TT-substituted templates in the presence of ADP. For comparison, the permanganate reactivity on the wt template (from Fig. 1) is also shown.