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Figure 4 | Breast Cancer Research

Figure 4

From: Leptin induces upregulation of sphingosine kinase 1 in oestrogen receptor-negative breast cancer via Src family kinase-mediated, janus kinase 2-independent pathway

Figure 4

JAK2 silencing or PI3K/Akt inhibition does not abrogate SK1 expression. (A-C) MDAMB-231 cells were transfected with specific siRNA against STAT3 (siSTAT3) and JAK2 (siJAK2) or control siRNA (siCont). Cells were starved overnight in serum-free media then exposed to 1,000 ng/ml leptin for 6 h. (D-F) MDAMB-231 cells were starved overnight in serum-free media then pre-treated with PI3K/Akt inhibitors wortmannin (1 μM) and LY294002 (10 μM) for 1 h followed by stimulation with 1,000 ng/ml leptin for 6 h. (A, D) Cell lysates obtained were separated on a 10% SDS-PAGE gel and probed for phosphorylation of STAT3, JAK2 and ERK1/2 to verify knockdown efficiency or Akt to confirm that the inhibitor worked under the experimental conditions. Expression of SK1 (B, E) and VEGF (C, F) determined by qRT-PCR, normalised against housekeeping genes (GAPDH, YWHAZ and UBC) and analysed using qBase software. Columns, mean of three independent experiments performed in triplicate; bars, SEM. (*, P <0.05; **, P <0.01; §, P <0.001; NS, not significant, P >0.05) when comparing levels of SK1, and VEGF mRNA to siCont levels.

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