Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: a Inset shows how the interaction of proteins A and B enables their SmBiT and LgBiT fusions (Promega, USA) to be brought into close proximity to each other, allowing complex formation to occur and reconstitution of the active NanoBit (Promega, USA) luciferase. Graph shows the reconstituted luminescence activity of the various combinations of potential interacting N- and C-terminal-fused SmBiT and LgBiT constructs linked either to full-length eIF4E or the eIF4G 604–646 fragment, respectively, co-transfected into HEK293 cells. Individual N- and C-terminal LgBiT-linked eIF4E and eIF4G constructs co-transfected with SmBiT-HALO served as negative controls. b Western blot analysis of HEK293 cells, either co-transfected with SmBiT-eIF4E and eIF4G 604–646 -LgBiT or with each construct alone in combination with empty vector DNA, in whole cell lysate (Left, WCL) and with m 7 GTP pulldown analysis (right). Mock control consisted of co-transfection of empty SmBiT and LgBiT vectors. eIF4E and SmBIT-eIF4E (highlighted with black arrows) or eIF4G 604–646 -LgBiT were detected by using anti-eIF4E and anti-NanoLuc antibodies. c SmBiT-eIF4E and eIF4G 604–646 -LgBiT constructs were either co-transfected together or separately co-transfected with corresponding empty vector DNA. Mock control consisted of co-transfection with both empty SmBiT and LgBiT vectors into HEK293 cells. d SmBiT-eIF4E was co-transfected with either eIF4G 604–646 -LgBiT or eIF4G 604–646(YLL) -LgBiT constructs. e SmBiT-eIF4E and eIF4G 604–646 -LgBiT constructs co-transfected into HEK293 cells with either empty vector (mock) or vectors containing 4EBP1 wild-type or 4EBP1 mutants (as indicated). f 4EGi1 and 4E1RCat were titrated onto HEK293 cell co-transfected with SmBIT-eIF4E or eIF4G 604–646 -LgBiT. Luciferase activity was measured after 4 h. Luminescence signals were normalised with those obtained from DMSO vehicle-treated cells. Compound titrations were also normalised with respect to the counter screen titrations of the compounds against full-length NanoLuc (Additional file : Figure S1). All values represent mean ± SD ( n = 3). The molecular mass of the protein marker is indicated in K d

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Luciferase, Activity Assay, Construct, Transfection, Western Blot, Plasmid Preparation, Control, Cotransfection, Marker

Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: a Titration of the dual MTORC1/2 active site inhibitor PP242 onto HEK293 cells co-transfected with the NanoBit eIF4E:eIF4G 604–646 system. b Western blot analysis of endogenous level of eIF4E, eIF4G and 4EBP1 in non-transfected 293FT extracts and associated m7GTP pulldowns of eIF4E containing complexes with differing treatment concentrations of PP242. c Titrations of the mTORC1 allosteric inhibitor Rapamycin and the potent mTORC1/2 active site inhibitor Torin onto HEK293 cells co-transfected with the NanoBit eIF4E:eIF4G 604–646 system. Western blot analysis of endogenous level of eIF4E, eIF4G and 4EBP1 in non-transfected 293FT extracts and associated m7GTP pulldowns of eIF4E containing complexes with differing concentrations of d Rapamycin or e Torin . f HEK293 cells were co-transfected with the NanoBit eIF4E:eIF4G 604–646 system together with either siRNA Ctrl (siCtrl) or si4EBP1 and PP242 titration was performed again as in a . The inset shows the level of 4EBP1 siRNA-mediated knockdown by western blot using anti-4EBP1 antibody. Beta-actin was visualised as loading control. IC 50 values were determined for the titrations described in sections a , c and f using four parameter curve fits. Non-linear regression analysis was performed in GraphPad (Prism). All values represent mean ± SD ( n = 3). The molecular mass of the protein marker is indicated in kilodaltons

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Titration, Transfection, Western Blot, Knockdown, Control, Marker

Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: a Western blot analysis of endogenous phosphorylation status of AKT and S6 kinases in non-transfected HEK293 cells treated with either PP242, Torin, Rapamycin or the MNK1/2 inhibitor CGP57380. b Western blot analysis of endogenous 4EBP1 phosphorylation status in non-transfected HEK293 cells treated with either the dual mTORC1/2 active site inhibitors PP242 and Torin, or the allosteric inhibitor of mTORC1 Rapamycin c PANC1 cells, which contain low levels of endogenous 4EBP1 were co-transfected with the NanoBit eIF4E:eIF4G 604–646 system with differing combinations of 4EBP1 mutant constructs with different sensitivities to mTORC1-mediated phosphorylation. Each transfection combination was normalised to its own DMSO treatment control. The molecular mass of the protein marker is indicated in kilodaltons

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Western Blot, Transfection, Mutagenesis, Construct, Control, Marker

Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: a HEK293 cells were transfected with either MOCK (empty vector), 4EBP1 WT (wild-type) or negative control 4EBP1 YLM construct. Cells were analysed 48 h post transfection for eIF4E S209 phosphorylation using the AlphaScreen Surefire assay. 4EBP1 constructs were also FLAG tagged. Inset western blot analysis of whole cell lysate derived from equivalent HEK293 cell transfections probed for phosphorylated eIF4E, endogenous 4EBP1, FLAG-tagged constructs and β-actin . b PC-3 cells transfected with the NanoBit eIF4E:eIF4G 606–646 system and treated with compound titrations of either the mTORC1/2 active site inhibitors, Torin and PP242, or the MNK1/2 kinase inhibitor CGP57380. c PC-3 cells were treated with titrations of either Torin, PP242 or CGP57380, and eIF4E phosphorylation was determined using the AlphaScreen Surefire eIF4E 209 phosphorylation assay. The signal was normalised to GAPDH levels, which were also determined using an AlphaScreen assay. d PANC-1 cells transfected with the NanoBit eIF4E:eIF4G 606–646 system and treated with compound titrations of either the mTORC1/2 active site inhibitors, Torin and PP242, or the MNK1/2 kinase inhibitor CGP57380. e PANC-1 cells were treated with titrations of either Torin, PP242 or CGP57380, and eIF4E phosphorylation was determined using the AlphaScreen Surefire eIF4E 209 phosphorylation assay. The signal was normalised to GAPDH levels, which were also determined using an AlphaScreen assay. f Table containing IC 50 values determined for eIF4E S209 phosphorylation and NanoBit eIF4E:4G disruption. IC 50 values were derived from fitting the relevant titration curves with four parameter models using non-linear regression (Prism, GraphPad Ltd.). All values represent mean ± SD ( n = 3). The molecular mass of the protein marker is indicated in kilodaltons

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Transfection, Plasmid Preparation, Negative Control, Construct, Amplified Luminescent Proximity Homogenous Assay, Western Blot, Derivative Assay, Phosphorylation Assay, Disruption, Titration, Marker

Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: HEK293 cells transfected with the NanoBit eIF4E:eIF4G 604–646 system and treated with compound titrations of either a the AKT inhibitors, Ipasertib and AZD5363, b the ERK1/2 inhibitors, Ulixertinib and SCH772984 or c the mTORC/PI3K dual inhibitor BEZ235 for 4 h, and then measured for luciferase activity. d Table displaying the IC 50 values determined for each compound titrated into the live cell NanoBit eIF4E:eIF4G 604–646 PPI system. e HEK293 cells were co-transfected with NanoBit eIF4E:eIF4G 604–646 system and either siRNA Ctrl or siRNA 4EBP1. Cells were then treated BEZ235 and luciferase activity assessed as in a , b and c . Four parameter models were fitted to the experimental data using non-linear regression to derive the IC 50 values (Prism, GraphPad Ltd.). All values represent mean ± SD ( n = 3). Data was normalised to DMSO controls

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Transfection, Luciferase, Activity Assay

Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: a HEK293 cells were transfected with the NanoBit eIF4E:eIF4G 606–646 system and treated either with titrations of the AKT inhibitor Ipasertib or the ERK 1/2 inhibitor SCH-772984. Additionally, transfected HEK293 cells were also treated with a titration of Ipasertib with SCH772984 present throughput at constant concentration of 1 μM. Data was normalised to DMSO controls and equivalent cell viability experiments. b Western blot analysis of selected titration points in a probed for TSC2, ERK, eIF4E S209 and 4EBP1 phosphorylation status (T37/T46). c Western blot analysis of a wider range of titration points in a using anti-phospho Serine 65 4EBP1 antibody. Beta-actin was used as loading control. See “ ”. d Transfected HEK293 cells were treated with either titrations of Ipasertib, the MNK 1/2 inhibitor CGP57380, or with titrations of Ipasertib with CGP57380 kept at different constant concentrations. Treatments were performed for 4 h and then transfected HEK293 cells were assayed for luciferase activity. All values represent mean ± SD ( n = 3). The molecular mass of the protein marker is indicated in kilodaltons

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Transfection, Titration, Concentration Assay, Western Blot, Control, Luciferase, Activity Assay, Marker

Journal: BMC Biology

Article Title: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

doi: 10.1186/s12915-019-0658-0

Figure Lengend Snippet: a Basal NanoBit eIF4E:eIF4G 606–646 luciferase activity in cells both co-transfected with GFP or GFP-Myc and the NanoBit eIF4E:eIF4G 606–646 system. b Western blot analysis of HEK293 cells co-transfected with the NanoBit eIF4E:eIF4G 606–646 system and either GFP or GFP-MYC post 48 h transfection. c HEK293 cells were co-transfected with the NanoBit eIF4E:eIF4G 606–646 system with either GFP or GFP-MYC. 48 h post-transfection HEK293 cells were titrated with BEZ235 and then assayed for luciferase after 4 h after treatment. d A bicistronic luciferase reporter, which measures the relative amount of cap-dependent translation (Renilla) to cap-independent translation (Firefly), was co-transfected with either empty vector (MOCK), GFP or GFP-Myc into HEK293 cells. Renilla and Firefly luciferase activity was measured 48 h post transfection and plotted as a ratio-metric value. The Renilla luciferase is under the control of a GC-rich 5′UTR whose activity correlated to cap-dependent translation, whilst the Firefly luciferase is regulated by an internal ribosomal site (IRES). e Western blot analysis of the BEZ235 titrations shown in c performed under identical co-transfection and treatment conditions (SE = short exposure, LE = long exposure). f Comparative plot of PANC1 and PC-3 cells co-transfected with the NanoBit eIF4E:eIF4G 606–646 system and treated with the dual mTOR/PI3K inhibitor BEZ235. Cells were assayed 4 h post treatment. g After 48 h co-transfection with NanoBit eIF4E:eIF4G 606–646 system and siRNA Ctrl or siRNA 4EBP1, PC-3 cells were treated with BEZ235 as for f and then assayed for luciferase activity. The level of 4EBP1 siRNA knockdown was demonstrated by western blot using anti-4EBP1 antibody. Beta-actin was visualised as a loading control. All values represent mean ± SD ( n = 3). The molecular mass of the protein marker is indicated in kilodaltons

Article Snippet: GAPDH and pS209 eIF4E levels were assessed according to the manufacturer’s instructions (PerkinElmer) for each sample.

Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Plasmid Preparation, Control, Cotransfection, Knockdown, Marker