Figure 7.

Proliferation of RAS-transformed cells is associated with elevated cytoplasmic HuR and requires down-regulation of AMPKα2 and CaMKKβ. (a) Immunoblot of cytoplasmic HuR levels in MEFs and NIH3T3 cells, without or with expression of HRAS^G12V. (b) Immunostaining of HuR in control or HRAS^G12V-transformed NIH3T3 cells, ± metformin treatment. (c) Ampka1 and Ampka2 mRNA levels in MEFs and NIH3T3 cells, ± HRAS^G12V. Transcript levels were determined by qRT-PCR. n=3 independent biological replicates, each sample assayed in triplicate; error bars represent S.D. Statistics were calculated using Student’s two tailed t test.; *p<0.05. (d) Immunoblot comparing CaMKKβ levels in MEFs and NIH3T3 cells, without or with expression of HRAS^G12V. (e) Proliferation of A549 cells over-expressing AMPKα2 and/or CaMKKβ. n=2 independent biological replicates, each time point assayed in triplicate; error bars represent S.E.M. Statistics were determined for day 6 using Student’s two tailed t test.; *p<0.05. (f) Immunoblot showing over-expression of AMPKα2 and CaMKKβ and CaMKKβ-dependent phosphorylation of AMPKα2 in A549 cells. Lysates from the cell populations described in panel (e) were analyzed on blots probed with the indicated antibodies. The anti-p-Thr172 (AMPKα) antibody recognizes modified forms of both AMPKα isoforms. (g) IF imaging of A549 cells expressing AMPKα2 and/or CaMKKβ. Cells were immunostained for HuR, or p-C/EBPβ and total C/EBPβ. (h) Quantitation of cytoplasmic HuR levels and p-C/EBPβ:total C/EBPβ ratios in the cells described in panel g. n=9 cells; error bars represent S.E.M. Statistical differences were determined by Student’s t test; *p<0.05.