Figure 1.
Western blot analysis of cytoplasmic(C) and nuclear(N) expression of PIM-2 in various cell lines.
Nuclear or cytoplasmic proteins (50 µg) were separated on a 15% SDS-PAGE. Blots were reacted with anti PIM-2 antibodies as primary antibody and HRP conjugated anti rabbit IgG secondary antibody. The membranes were stripped twice and reacted once with anti RCC1 antibody as control for nuclear proteins and once with anti β-tubulin as control for cytoplasmic proteins.
Figure 2.
Over-expression of the 34 kDa isoform of the human PIM-2 protein in HeLa cells.
(A) Nuclear and cytoplasmic protein extracts (50 µg) from cells over-expressing HA-PIM-2 (Pim-2), control cells transfected with a HA vector (HA) or untreated control cells (HeLa), were analyzed by Western blotting using antibodies against either the PIM-2 protein or the HA-tag. For equal loading reference, the blot was stripped and re-probed with antibodies against β-actin (cytoplasmic fractions) or RCC1 (nuclear fractions). (B) Immunocytochemical analysis of HeLa cells transiently transfected with the HA-Pim-2 encoding plasmid, using either rabbit anti-PIM-2 antibodies (PIM-2) or mouse anti-HA antibodies (HA). Alexa 488 conjugated anti-rabbit (green) and Alexa 594 conjugated anti-mouse (red) secondary antibodies were used for PIM-2 and HA detection, respectively. In right lower panel PIM-2 and HA signals are merged. Nuclei were stained with Dapi (blue). Bar represents 10 µm. (C) Total protein extracts (50 µg) from cells over-expressing the 34 kDa isoform of PIM-2 tagged with the HA-tag at its N-terminus and with the Myc-His-tag at its C-terminus (HA-PIM-2-Myc), or from untreated control cells (HeLa), were analyzed by Western blotting, using antibodies against the PIM-2 protein (PIM-2). The blot was stripped and re-probed with antibodies against the Myc-tag (Myc), stripped again and re-probed with antibodies against the HA-tag. For equal loading reference, the blot was stripped once again and re-probed with antibodies against tubulin.
Figure 3.
PIM-2's overexpression in HeLa cells promotes G1 arrest and increases apoptosis.
(A) Upper panel - Light microscope images (×40) of cells 48 hours after transfecting equal amounts of cells with either HA-Pim-2 vector (pHA-Pim2) or empty control vector (pHA-empty), and under identical culture conditions. Middle panel – Western blot analysis, using anti HA antibodies, showing the relative amounts of either HA-PIM-2 or HA-PIM-2 KD in the tested cells. Lower panel - Fold increase in cell number calculated 48 hours after transfecting equal amounts of cells with either the HA-Pim2 plasmid (Pim-2), an empty HA control vector (HA) or with a kinase dead form of Pim-2 (PimKD). Data shown are the average of four independent experiments. Asterisk represents statistically significant differences (p<0.05). (B) FACS analysis of cell cycle distribution of PI stained cells 48 hours after transfection with either HA-Pim2 plasmid (Pim-2), HA-Pim2 kinase-dead plasmid (Pim-2 KD), control empty vector (HA) or untreated cells (HeLa). (C) A comparison between the average percentage of cells (four independent experiments) at the sub-G1 and G1 phases, in Pim-2 expressing cells versus HA control cells (Asterisks represents statistically significant differences p<0.014 and p<0.007, respectively).
Figure 4.
p57 expression and T14/Y15 phosphorylation on CDK2 in HeLa cells over-expressing the 34 kDa PIM-2 isoform.
(A) RT-PCR analysis of p57 transcripts in control cells (HA control) and in Pim2 over-expressing cells (HA Pim-2). Actin specific primers were used as reference for equal loading. Western blot analysis was used to evaluate the p57 protein in nuclear extracts (40 µg) from HA control cells and from cells over-expressing Pim-2 (HA Pim-2). Anti-RCC1 antibodies were used as reference for equal loading. The average relative level of p57 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of p57 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of p57 in control cells was determined as 1. Differences were just above the statistically significance of p<0.05. (B) Western blot analysis of phosphorylated CDK2 on T14/Y15 (pCDK2) in nuclear extracts from cells over-expressing Pim-2 (HA Pim-2) and HA control cells, using phospho T14/Y15 specific antibodies. Blots were stripped and reprobed once with CDK2 specific antibodies and then once again with RCC1 antibodies as reference for equal loading. The average relative level of pCDK2 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of pCDK2 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of pCDK2 in control cells was determined as 1. Asterisk represents statistically significant differences (p<0.04) (C) Western blot analysis of pCDK2 in total protein extracts (40 µg) from Pim-2 silenced cells, via siRNA (si-Pim), and from cells transfected with scrambled control siRNAs (si-control). Antibodies specific to β-actin were used as reference for equal loading.
Figure 5.
Down-regulation of CDC25A expression in HeLa cells over-expressing the 34 kDa form of PIM-2.
(A) Western blot analysis of the CDC25A protein in nuclear extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells transfected with empty HA vector (HA). Anti-RCC1 antibodies were used as reference for equal loading. The average relative level of CDC25A in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of CDC25A signals normalized according to the RCC1 signal (average of three independent experiments). Average level of CDC25A in control cells was determined as 1. Asterisk represents statistically significant differences (p<0.05). (B) RT-PCR analysis of Cdc25A transcripts in Pim2 over-expressing cells (Pim-2) and in control cells (HA). Actin specific primers were used as reference for equal loading. (C) PIM-2 promotes CDC25A degradation via the proteasome. Western blot analyses of β-catenin (top panel), as control for proteasomal inhibition, and CDC25A (botom panel), were performed on total protein extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells (HA) after treatment with the proteasome inhibitor, MG-132, for 0, 2 or 5 hours (h). Antibodies specific to tubulin were used as a reference for equal loading. (D) PIM-2 directly phosphorylates CDC25A in an in-vitro kinase assay. 293 cells were transfected (+), or not (−), with a Flag-tagged CDC25A expressing vector, and the tagged protein was immunoprecipitated using anti Flag antibodies. The immunoprecipitated protein was used as a substrate in a PIM-2 kinase assay (lower panel). Expression of the tagged protein was verified by Western analysis (upper panel), and equal loading was verified by stripping the blot and probing it with anti tubulin antibodies (middle panel).
Figure 6.
Up-regulation of E2F-1 and p73 expression in HeLa cells over-expressing the 34 kDa form of PIM-2.
(A) RT-PCR analysis of E2F-1 transcripts in Pim2 over-expressing cells (Pim-2) and in control cells (HA). Actin specific primers were used as reference for equal loading. (B) Western blot analysis of the E2F-1 protein in nuclear extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells with empty HA vector (HA). Blot was stripped and reprobed with antibodies specific to RCC1 as reference for equal loading. The average relative level of E2F-1 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is shown in the right panel. Comparison was based on densitometric analysis of E2F-1 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of E2F-1 in control cells was determined as 1. Asterisk represents statistically significant differences (p<0.01). (C) RT-PCR analysis of p73 transcripts in Pim2 over-expressing cells (Pim-2) and in control cells (HA). Actin specific primers were used as a reference for equal loading. The average relative level of p73 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is shown in the right panel. Comparison was based on densitometric analysis of p73 signals normalized according to the β-actin signal (average of three independent experiments). Average level of p73 in control cells was determined as 1. Differences were statistically significant (p<0.01). (D) Western blot analysis of the p73 protein in total cell extracts (50 µg) from cells over-expressing Pim-2 (Pim-2) and control cells with empty HA vector (HA). Blot was stripped and reprobed with antibodies specific to β-tubulin as reference for equal loading.
Figure 7.
Dominant negative(DN) p73 or p73 silencing reverse the PIM-2 effect on cells over-expressing the 34 kDa PIM-2 isoform.
(A) Light microscopic view (×40) of cells 48 hours after transfecting equal amounts of cells with an empty HA vector as control (control), HA-Pim2 plasmid alone (Pim-2) and a HA-Pim-2 plasmid together with a dominant negative form of p73 (Pim-2+p73DN), and under identical culture conditions. (B) Percent of sub-G1 apoptotic cells, and (C) percent of cells in the G1 phase of the cell cycle, in the specified cultures, as revealed by FACS analysis. Results represent an average of four independent experiments. Asterisk represents statistical significance p<0.01. (D) Western analysis showing siRNA-silencing of p73 compared to untreated control or control of cell treated with scrambled siRNA. (E) FACS analysis of cell cycle pattern of PI stained cells to which siRNAs were introduced [control scrambled (scr.) or p73-directed siRNA (p73)] 24 h prior to transfection with either empty HA plasmid (HA+scr. or HA+73) or HA-Pim-2 plasmid (PIM-2+scr. or PIM-2+73). FACS analysis was performed 76 h after transfection with plasmids. Horizontal line in each pattern indicates the channels that were included in calculation of the sub-G1 phase. (F) Percent of cells at the sub-G1 phase calculated from the cell cycle patterns presented in panel E.
Figure 8.
A proposed model explaining the molecular pathway through which PIM-2 exerts G1-arrest and promotes apoptosis.