Fig 2: eIF4A inhibition changes the polysomal association of certain mRNAs.Cultured cells (SH-SY5Y, N2a or HeLa) were treated with 10 µM hippuristanol (+) or DMSO control (-) for 10 minutes, lysed and mRNA fractionated on a 10%–50% sucrose gradient. OD260nm absorbance was determined with simultaneous 1 ml fraction collection to determine subpolysomal and polysomal fractions (fraction number and subpolysomal or polysomal association indicated). Northern analysis was performed to determine the positions of mRNAs for: A Actin and PABP which do not change in polysome associations; B APP and tau which become less polysomally associated; C TXN, NDUFB2 and SOD1 which become more polysomally associated. Relative levels of RNA are indicated by dark grey (DMSO control) and light grey (10 µM hippuristanol) bars beneath each fraction, as percentages of the total amount RNA in each gradient.

Fig 3: CD40-mediated apoptosis in carcinoma cells. Schematic diagram of the signalling pathway that underpins mCD40L-mediated, tumour cell-specific apoptosis. Unlike CD40 engagement by soluble agonist that does not adequately activate the receptor to stabilise TRAF3 and initiate signalling, ligation of CD40 by mCD40L recruits and upregulates TRAF3 and activates p40phox to induce rapid ROS elevation – which appears to be essential for TRAF3 stabilisation. The generated ROS mediates the oxidation and inactivation of Trx (Trx-1), permitting activation of ASK-1 and subsequent phosphorylation of MKK4 and JNK to activate the JNK/AP-1 cascade which is responsible for the induction of Bax and Bak expression. This triggers the intrinsic, cytochrome c-driven, caspase-9 dependent mitochondrial pathway that drives apoptosis.

Fig 4: mRNA levels do not account for the change in protein levels following hippuristanol treatment.Northern analysis 4 or 24 hours following 10 minutes 10 µM hippuristanol treatment, using probes specific to APP, TAU, Actin and TXN, indicates no significant decrease in these mRNA levels. Mean relative proportions of RNA with standard errors are indicated in the charts beneath each gel image.

Fig 5: Expression of proteins associated with AD is altered by eIF4A inhibition. A Western analysis was performed on lysates from SH-SY5Y cells treated with 10 µM hippuristanol (+) or DMSO control (-) for 4 or 24 hours with antibodies specific to APP, TAU and TXN (bands of around 70 kDa, 60 kDa and 12–14 kDa respectively). The 70 kDa band was detected by the APP specific antibody representing a cleavage product specific to K+ isoforms [53]. Secreted A-beta was measured by western analysis of cell culture media. The 56 kDa product is interpreted as an aggregate of A-beta, as has previously been reported for western analysis of brain tissue [51]. Protein levels were normalized to actin, and mean relative proportions of protein with standard errors are indicated beneath each gel image. Significant differences (p=0.05 by t-test) are indicated by asterisks. B. Immunoprecipitation following 35S-methionine labeling demonstrates a reduction in novel synthesis of APP, TAU but not TXN when SH-SY5Y cells are treated with 10 µM hippuristanol. Secreted A-beta levels are also reduced, as determined by immunoprecipitation from cell culture medium. Mean relative proportions of protein with standard errors are indicated beneath each gel image. Significant differences (p=0.05 by t-test) are indicated by asterisks. C. Secreted A-beta levels in the medium of 9×105 SH-SY5Y cells were assayed by ELISA 24 hours following treatment with 10 µM hippuristanol (+), demonstrating a modest but reproducible decrease in comparison to control cells (-). Means and standard errors of 6 replicates are shown p = 0.05.