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HeLa cells do not express LKB1 (LKB1 -/-), so genetically modified HeLa cells were utilised to examine how LKB1 expression/activity could influence the cell cycle. These cells either expressed FLAG-tagged wild type-LKB1 (WT-LKB1) or kinase dead-LKB1 (KD-LKB1) under the control of a tetracycline inducible promoter (Sapkota et al.,2002). Both constructs could be induced by the

incubation with tetracycline although expression of KD-LKB1 was lower than that of WT-LKB1 (Figure 5-1). As anticipated in LKB1 -/- HeLa cells, AICAR (1 mM, 1 h) caused an increase in phospho-S79 ACC but did not alter phospho-T172 AMPK indicating the allosteric effect of AICAR/ZMP on AMPK activity (Figure 5-1). Similarly, in KD-LKB1 expressing cells, AICAR stimulated phosphorylation of ACC but not AMPK (Figure 5-1). WT-LKB1 HeLa cells had higher basal and AICAR- stimulated phosphorylation of ACC, an increase in phospho-T172 AMPK was also apparent following stimulation with AICAR as expected (Figure 5-1).

Next the subcellular localisation of LKB1 in these cells was investigated by immunofluorescence microscopy. Staining of LKB1 -/- HeLa cells with anti-LKB1 antibodies produced low levels of non-specific immunoreactivity (Figure 5-1). Higher levels of LKB1 immunoreactivity were detectable in both LKB1-WT and LKB1-KD HeLa cells, which localised to the cytoplasm and nucleus (Figure 5-1). LKB1 also localised to the intracellular bridge but not to the midbody.

156 Figure 5-1. Subcellular localisation of LKB1 during telophase

(A) KD-LKB1 or WT-LKB1 HeLa cells were incubated in the presence or absence of tetracycline (1 μg/ml) for 24 h to induce expression of FLAG-tagged mutant LKB1. KD-LKB1, WT-LKB1 and LKB1 -/- HeLa cells were subsequently incubated in the presence or absence of AICAR (1 mM, 1 h) and lysates prepared. Lysates were resolved by SDS-PAGE and subjected to immunoblotting with the antibodies indicated, a HUVEC lysate was included as a positive control. N=1. (B) Quantification of tetracycline-induced LKB1 expression relative to GAPDH. Presented as %WT-LKB1 expression. N=4. (C-E) KD-LKB or WT-LKB1 HeLa cells were incubated with tetracycline (1 μg/ml) for 24 h. LKB1 -/-, KD-LKB1 or WT-LKB1 HeLa cells were fixed with 4% (w/v) PFA and stained with anti- tubulin (Abcam, ab6160) and anti-LKB1 (New England Biolabs, #3050) antibodies. DNA was stained with RedDot2. Scale bar=10 μm. Representative images are shown from three independent experiments in each case.

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As a genetic interactions were described between the AMPK kinase ssp1 and both

ark1 and plo1 in Chapter 4, the localisation of their mammalian orthologues

(aurora A/aurora B and PLK1) was assessed in WT-LKB1, KD-LKB1 HeLa cells, LKB1 -/- Hela cells and HUVECs. Aurora A immunoreactivity was observed in the cytoplasm and nuclei of all tested cell lines (Figure 5-2). Midbody localisation of aurora A was only observed in HUVECs (Figure 5-2). In HeLa cells, there was enrichment of aurora A at the intracellular bridge but not at the midbody proper (Figure 5-2). Similarly, aurora B immunoreactivity was detected in the cytoplasm and nuclei of LKB1 -/- HeLa cells, LKB1-KD HeLa cells, LKB1-WT HeLa cells and HUVECs (Figure 5-3). Midbody localisation of aurora B was only observed in HUVECs (Figure 5-3). Finally, PLK1 immunoreactivity was detected in the

cytoplasm and nuclei of all tested cells and at the midbody in HUVECs (Figure 5- 4).

158 Figure 5-2. Subcellular localisation of aurora A during telophase

(A) LKB1 -/- HeLa cells, (B) KD-LKB1 HeLa cells, (C) WT-LKB1 HeLa cells and (D) HUVECs were fixed with 4% (w/v) PFA. Cells were stained with anti-tubulin (Abcam, ab6160) and anti-aurora A (Abcam, ab1287) antibodies. DNA was stained with RedDot2. KD-LKB1 and WT-LKB1 HeLa cells were pre-treated with tetracycline (1 μg/ml) for 24 h. Scale bar=10 μm. Representative images are shown from three independent experiments in each case.

159 Figure 5-3. Subcellular localisation of aurora B during telophase

(A) LKB1 -/- HeLa cells, (B) KD-LKB1 HeLa cells, (C) WT-LKB1 HeLa cells and (D) HUVECs were fixed with 4% (w/v) PFA. Cells were stained with anti-tubulin (Abcam, ab6160) and anti-aurora B (Abcam, ab2254) antibodies. DNA was stained with RedDot2. KD-LKB1 and WT-LKB1 HeLa cells were pre-treated with tetracycline (1 μg/ml) for 24 h. Scale bar 10=μm. Representative images are shown from three independent experiments in each case.

160 Figure 5-4. Subcellular localisation of PLK1 during telophase

(A) LKB1 -/- HeLa cells, (B) KD-LKB1 HeLa cells, (C) WT-LKB1 HeLa cells and (D) HUVECs were fixed with 4% (w/v) PFA. Cells were stained with anti-tubulin (Abcam, ab6160) and anti-PLK1 (Novus Biologicals, NB-100-547) antibodies. DNA was stained with RedDot2. KD-LKB1 and WT- LKB1 HeLa cells were pre-treated with tetracycline (1 μg/ml) for 24 h. Scale bar 10=μm.

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Next, the expression of WT-LKB1 or KD-LKB1 on HeLa cell polyploidy was

assessed. Cells were incubated with AMPK activators or inhibitors for 18 h prior to staining with PI and assessment of DNA content by flow cytometry. Expression of either WT-LKB1 or KD-LKB1 did not influence HeLa cell DNA content (Figure 5- 5). Compound C (10 µM) caused a significant reduction in the proportion of KD- LKB1 cells with 2n DNA coinciding with an increase in the proportion of cells with 4n DNA (Figure 5-5). In contrast, compound C did not significantly alter DNA content of WT-LKB1 HeLa cells (Figure 5-5). Incubation with STO-609, A769662, AICAR or canagliflozin did not alter the DNA content of either KD-LKB1 or WT- LKB1 HeLa cells.

HeLa cells which expressed KD-LKB1 had low basal levels of phospho-S79 ACC which did not increase in response to either AICAR, A769662 or canagliflozin (Figure 5-5I). A769662 and AICAR significantly increased phospho-S79 ACC in WT- LKB1 HeLa cells, however, canagliflozin did not stimulate AMPK activity. Neither STO-609 nor compound C reduced basal phospho-S79 ACC levels in either cell type (Figure 5-5I). Expression of LKB1 was markedly lower in KD-LKB1 expressing cells. Surprisingly, both compound C and AICAR significantly increased levels of WT-LKB1 relative to GAPDH (Figure 5-6). AICAR stimulated an increase in phospho-T172 AMPK in WT-LKB1 HeLa cells but not in KD-LKB1 HeLa cells, no other treatments altered phospho-T172 AMPK (Figure 5-6).

164 Figure 5-5. The effect of modulating AMPK activity on the DNA content of WT- and KD-LKB1 expressing HeLa cells

KD-LKB1 and WT-LKB1 inducible HeLa cells were pre-treated with tetracycline (1 μg/ml) for 24 h. HeLa cells were then seeded into 6-well plate and incubated for 4 h until cells adhered. Cells were subsequently treated as indicated for 18 h with tetracycline (1 μg/ml). (A) Representative scatter plots and histogram overlays comparing DMSO (blue) with activator/inhibitor treated cells (pink) of WT-LKB1 HeLa cells. (B) Representative scatter plots and histogram overlays comparing DMSO (blue) with activator/inhibitor treated cells (pink) of KD-LKB1 HeLa cells. (C) Comparison of WT- LKB1 (green) and KD-LKB1 (blue) HeLa histograms. (D-G) Analysis of the proportion of cells with 2n, 2-4n, 4n or >4n DNA content presented as % cells ± SEM from 3 independent experiments. (H) HeLa cells were treated with the indicated compounds for 18 h and lysates prepared. Lysates were resolved by SDS-PAGE and immunoblotted using the antibodies indicated. Representative images are shown. (I) Quantification of phospho-S79 ACC relative to total ACC, presented as % DMSO of WT-LKB1 ± SEM. (J) Quantification of LKB1 relative to GAPDH, presented as % DMSO of WT- LKB1. (K) Quantification of phospho-T172 AMPK relative to total AMPK, presented as % DMSO of WT-LKB1. * p<0.05, **p<0.01 relative to cells incubated with DMSO and # p<0.05 as indicated (two-way ANOVA). N=3.

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