Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: a X-ray crystal structure of eIF4E (PDB: 5T46 ) overlaid with a protein binding partner (eIF4G peptide). Protein secondary structure is represented as coloured ribbons, with eIF4E (yellow), and eIF4G peptide (green). The Connolly surface of m7-GDP is displayed in magenta. b Protein surface of eIF4E showing the canonical binding region as a Connolly surface coloured by hydrophobicity, with red indicating a strongly hydrophobic (lipophilic) region. A fragment of 4E-BP1 is overlaid in ribbon representation (cyan). Residues highlighted (single letter codes) form part of the consensus sequence (YXXXXLφ) for the canonical binding partners, in this case 4E-BP1 (PDB: 3U7X ) . Where Y = Y54, X = any residue, L = L59, φ = lipophilic residue corresponding to M60 for 4E-BP1. c Protein surface of 4E-BP1-eIF4E fusion protein as a Connolly surface coloured by hydrophobicity.

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Protein Binding, Binding Assay, Sequencing, Residue

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: The secondary structure of eIF4E is shown in ribbon representation. The majority of the protein (grey), canonical peptide derived from 4E-BP1 attached to the N-terminus of eIF4E (blue), loop region between the C-terminus of the α1-helix and N-terminus of the β3-sheet (yellow). The ligand is depicted as green lines. Sidechains from protein residues within 4 Å of the bound ligand are highlighted in orange with single letter codes. Hydrogen bonds between ligand, protein and water are denoted with black dashed lines. Key waters are shown as red spheres and labelled W1 – W4. a Compound 1 bound in site 2 (resolution 1.85 Å). Key features are highlighted including the location of site 2 in comparison to the Cap binding site (the Cap-site ligand (m7-GTP) is shown for illustration purposes only and was not included during protein purification or the screening process). The surface representations of 1 and m7-GTP are shown in cyan and magenta respectively. b Magnified view of binding site 2 with compound 1 bound which has been rotated anti-clockwise by 90°. c Compound 2 structure (resolution 1.89 Å). d Compound 3 structure (resolution 1.93 Å). e Compound 4 structure (resolution 1.97 Å).

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Derivative Assay, Comparison, Binding Assay, Protein Purification

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: Biophysical and biochemical data for site 2 binders

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Inhibition

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: Key residues from eIF4E (black) or eIF4G peptide (red italics) are shown as single letter codes . a Compound 4 structure showing the protein Connolly surface coloured by hydrophobicity, red indicating strongly hydrophobic areas. As the site is highly enclosed, the surface associated with W76 and Q81 has been removed to improve visualisation. b Compound 4 structure showing the protein Connolly surface coloured by hydrophobicity and the ligand Connolly surface (yellow). c Overlay of eIF4G peptide (green ribbon) with the protein conformation of compound 4 bound eIF4E (grey ribbon), the reorganised α1-helix and flexible loop region is displayed as a yellow ribbon, the Connolly surface of compound 4 is displayed in cyan. The original loop conformation from the 5T46 structure of eIF4E bound to an eIF4G peptide is displayed as a red ribbon. d Overlay of eIF4G peptide (green ribbon and green Connolly surface) with the protein conformation of compound 4 bound eIF4E. The orientation has been rotated by ~ 90° in the horizontal plane compared to that shown in Figure 3c.

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques:

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: a Lysates from SW620 cells were incubated with 1–100 µM compound 4 or 5 or positive control peptide (RIIY) for 30 min. Endogenous eIF4E was immunoprecipitated and immunoblotted for eIF4G, 4E-BP1 and eIF4E. Quantitation of 4E-BP1 ( b ) or eIF4G ( c ) with endogenous eIF4E in SW620 and HeLa cell lysates, determined by the electro-chemiluminescent binding assay following incubation for 30 min with DMSO vehicle (Cont), 100 µM compound 4 or 100 µM RIIY peptide. Complexes were immobilised by an eIF4E antibody and captured eIF4E, eIF4G and 4E-BP1 detected by their respective secondary antibodies. Values represent ratios of 4E-BP1:eIF4E or eIF4G:eIF4E electro-chemiluminescence relative to DMSO control ( n = 2 biological replicates). d Electro-chemiluminescent assay for binding of eIF4G or 4E-BP1 with eIF4E in SW620 (n = 2 biological replicates), or ( e ) in HeLa lysates ( n = 3 biological replicates, mean ± SD) following incubation for 30 min with compound 4 or 5 . Results are expressed as luminescence signals relative to DMSO control. f Quantification of eIF4E:eIF4G interaction in H1299 cells by electro-chemiluminescent assay. Cell lysates treated with RIIY 4E-BP1 derived peptide or RIIG negative control peptide at 0.1–100 µM for 30 min ( n = 2 biological replicates). g Quantification of the endogenous eIF4E:eIF4G interaction in H1299 cell lysates at 0.1–100 µM (for 6 h) of compound 4 or 5 , as measured by electro-chemiluminescent assay (mean ± SD from n = 3 biological replicates). h HeLa cell lysates for in vitro translation were incubated for 30 min with 1, 10, 100 µM of compound 4 or 5 . Results are expressed as firefly or renilla luminescence signal normalized to DMSO control and expressed as % (mean ± SD from n = 3 biological replicates). Significance was determined using two-sided unpaired t-test comparing compound 4 to compound 5 at each concentration. Statistically significant p -values ( p < 0.05) are shown on the plot and source data is located in the Source Data file.

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Incubation, Positive Control, Immunoprecipitation, Quantitation Assay, Binding Assay, Control, Derivative Assay, Negative Control, In Vitro, Concentration Assay

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: a Top: Representative immunoblot ( n = 2 biological repeats) showing eIF4E protein stabilisation in H1299 cells at 57.6 °C following compound treatment. Cells were treated with compound 4 or compound 5 (50 μM) for 6 hrs and then incubated at 57.6 °C. Bottom: Representative immunoblot ( n = 3 biological repeats) following treatment with compound 4 (10, 5, 1, 0.5 μM) or compound 5 (10 μM) for 6 hrs followed by incubation at 57.6 °C. Vinculin was used for loading control. b eIF4E stabilisation in H1299 cells at 57.6 °C following compound 4 treatment was quantified from immunoblots using Image J and normalised to vinculin control. Statistically significant p values ( p < 0.05) are shown on the plot (mean ± SEM from n = 3 biological replicates). c Quantification of the endogenous eIF4E:eIF4G interaction in intact H1299 cells treated at 100 µM for 6 or 16 hr (mean ± SD from n = 3 biological replicates). Statistically significant p -values ( p < 0.05) are shown on the plot. d Cells were transfected with a protein synthesis reporter expressing a bicistonic mRNA with a cap-dependent luciferase reporter (fLuc) and a cap-independent luciferase (rLuc) driven by a viral IRES and were treated with compound 4, 5 (10 or 25 µM) or cycloheximide (CHX; 100 µM) for 24 hr. Luminescence was measured using the Dual-glo luciferase assay. All p values for compounds 4 and 5 were not statistically significant ( p > 0.05; mean ± SEM from n = 3 biological replicates). e Cell viability measured by cell titre blue assay in cells treated with compound 4 or 5 for 4 days (mean ± SEM from n = 3 biological replicates). For ( b – d ) significance ( p < 0.05) was determined using an ordinary one-way ANOVA with Tukey multiple comparisons test, comparing compound 4 treatment with the control or negative control compound 5 treatment conditions. Source data is located in the Source Data file.

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Western Blot, Incubation, Control, Transfection, Expressing, Luciferase, Negative Control

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: a Crystal structure of eIF4E bound to a 35-residue fragment of eIF4G (PDB: 5T46 ) showing key features previously described in Figs. a and . Highlighted in cyan are the residues selected for mutational analysis which gave suitable expression levels (W56, W73, L85, L134 and S209). b Quantification of eIF4E:eIF4G interaction determined by the electro-chemiluminescent assay in cells transfected with wild type (WT) eIF4E or a series of eIF4E mutants. c Quantification of N-terminal FLAG-tagged eIF4E expression in cells transfected with wild type (WT) eIF4E or a series of eIF4E mutants. d Quantification of eIF4G normalised to eIF4E (data from b and c ) in cells transfected with wild type (WT) eIF4E or a series of eIF4E mutants. All plots ( b – d ) show mean ± SD from n = 3 biological replicates with significance for each mutant relative to the WT control using an ordinary one-way ANOVA with Tukey multiple comparisons test. Significant p -values ( p < 0.05) are shown on the plots and the source data is located in the Source Data file.

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Residue, Expressing, Transfection, Mutagenesis, Control

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: a Schematic of the generation of stable dTAG eIF4E single clones in H1299 cells for eIF4E degradation (created in BioRender. Powers, M. (2024) https://BioRender.com/t36s392 ). A stable cell pool expressing eIF4E-dTAG was established, endogenous eIF4E was removed using CRISPR/Cas9, and single cell clones were isolated. Treatment of isolated clones with the heterobifunctional dTAG V −1 molecule recruits E3 ligase VHL to degrade eIF4E-dTAG. Four eIF4E-dTAG CRISPR clones were selected: two C-terminal FLAG-tagged (C2-2, C3−1) and two N-terminal FLAG-tagged (N2−1, N3-2). b Immunoblot analysis of eIF4E, FLAG-tagged eIF4E, and MCL1 expression. Parental H1299 cells and the selected eIF4E-dTAG clones were treated with 0.5 and 1 µM dTAG V −1 for 6, 8, or 16 h. Vinculin was used as a loading control ( n = 3 biological replicates). c Real-time cell growth measurements of the selected eIF4E-dTAG clones treated with 0.5 or 1 µM dTAG V −1. Cell confluency (%) was monitored every 4 h over 6 days using Incucyte Zoom. Significance was determined using a one-way ANOVA with Tukey multiple comparisons test comparing dTAG V −1 treatment with the control in each cell line. Significant p values ( p < 0.05) are shown (mean ± SEM from n = 3 biological replicates), the source data is located in the Source Data file. d Parent H1299 cells were treated with eIF4E siRNA (1 µg; eIF4E siRNA) or control siTOOLs (PAR) for 72 h ( n = 3 biological replicates). The four eIF4E-dTAG clones were treated with vehicle (Con no dTAG) or 500 nM dTAG V −1 (dTAG) for 72 h ( n = 1 repeat from each individual clone) and global proteomes profiled. Significant differences between control and treated samples were determined using MSstats. Plots show signal intensity data for eIF4E (CON no dTAG v dTAG and eIF4E siRNA v PAR both p adj < 0.05) and MCL1 (p adj > 0.05; not significant). e Log 2 [signal intensity] for proteome profiles of the individual eIF4E-dTAG clones with the control parent H1299 cells. Each point represents the ratio of individual protein expression between the control parent and different control dTAG clones (C2-2, C3-1, N2−1 and N3-3). Diagonal lines represent a 1.5-fold change in protein expression (Supplementary Data ).

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Clone Assay, Stable Transfection, Expressing, CRISPR, Isolation, Western Blot, Control

Journal: Nature Communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: eIF4E dTAG C3-1 clone or C3−1 were transfected with wild-type eIF4E (WT) or a series of eIF4E mutants and treated with 0.5 or 1 µM dTAG V -1. a Quantification of MCL1 immunoblot signal using Image J. Results are expressed as % MCL1 normalized to vinculin (mean ± SEM from n = 3 biological replicates) following 6 hr dTAG V −1 treatment (Supplementary Fig. ). One sample t and Wilcoxon tests (two-sided), using 100% as hypothetical control value, were performed and compared with the control in each cell line. b , c Real-time cell growth measurements monitored every 4 h over a 6-day period. Cell confluency (%) was calculated using Incucyte Zoom software and (mean ± SEM n = 3 biological replicates). Ordinary one-way ANOVA with Tukey multiple comparisons test compared treatment with the control in each cell line. Significant p -values (< 0.05) are shown on the plots. d Representative immunoblot of 3 biological replicates of eIF4E and MCL1 protein expression following 16 hr treatment of dTAG V -1 (1 µM) or compound 4 (25 μM) or in combination in H1299 parental cells, the eIF4E dTAG C3-1 clone or C3-1 transfected with W73F or L85R mutant. Vinculin was used as loading control. H1299 (PAR) cell lines were treated with 1 μM dTAG V -1 only as qualitative control. Vinculin was used as loading control. Quantification from MCL1 immunoblots normalised to control is shown in Supplementary Fig. . e Real-time cell growth of the eIF4E dTAG C3-1 clone and C3-1 transfected with W73F or L85R mutant following treatment with 25 µM compound 4 ± 1 µM dTAG V -1 were monitored and analysed as described for ( c ). Signifi c ant p -values (< 0.05) are shown on the plots and the source data is located in the Source Data file.

Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat# MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20 μg used to coat 1 ml streptavidin magnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.

Techniques: Transfection, Western Blot, Control, Software, Expressing, Mutagenesis

Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: a Comparing the protein expression of a panel of translation regulators between tamoxifen sensitive (MCF-7 and ZR-75) and resistant cells (LCC2 and AK-47). Western blotting was used to determine the expression of the indicated protein candidates. Tubulin was used as the loading control. b Overexpression of eIF4E in MCF-7 and ZR-75 breast cancer cells could induce distinctive molecular alterations in polysomes. The cells were transfected with pCMV6_eIF4E for 72 h. RNA sequencing was performed to determine the effect of eIF4E overexpression on mRNA profile in the polysome. Heatmap was used to show the profiles of the molecular features. c Top 10 molecular pathways being enriched by overexpression of eIF4E. KEGG pathway analysis was performed to identify pathways which were potentially enriched in eIF4E overexpressing breast cancer cells. d The effect of eIF4E overexpression on the mRNA levels of eIF4E, ERα and FOXM1 in MCF-7, ZR-75, LCC2 and AK-47 cells. qPCR was performed to determine the levels of the corresponding mRNAs. Actin was used as the internal control. Results were expressed as mean ± s.d. from three independent experiments. Student t- test was employed. *** represents P < 0.001. e The effect of eIF4E overexpression on the protein levels of eIF4E, ERα and FOXM1 in MCF-7, ZR-75, LCC2 and AK-47 cells. The cells were transfected with 2 µg of pCMV6_eIF4E (eIF4E O/E) or pCMV6 (Ctrl O/E). Whole cell lysates were harvested after 72 h posttransfection. Western blot was performed. Tubulin was used as the loading control.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Expressing, Western Blot, Over Expression, Transfection, RNA Sequencing Assay

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: Polysome fractionation was performed on ( a ) MCF-7 and ( b ) ZR-75 cells. Overexpression of eIF4E was mediated by transfection of 4 μg of pCMV6_eIF4E while knockdown of eIF4E was mediated by transfection of 120 pmol of eIF4E siRNA#1. Cells were harvested after 72 h posttransfection. The absorbance at 260 nm in each of the fractions was monitored. Fractions 13–17 were regarded as the light fractions while fractions 19–23 were regarded as heavy fractions (polysome). Ctrl represents untreated sample, eiF4E KD represents eIF4E knockdown, eIF4E OE represents eIF4E overexpression. c Alteration of eIF4E expression could alter the abundance of ERα and FOXM1 mRNAs in the heavy fractions in MCF-7 and ZR-75. d Alteration of eIF4E expression did not affect the abundance of ERα and FOXM1 mRNAs in the light fractions in MCF-7 and ZR-75. qPCR was performed to detect the presence of the mRNAs in the heavy and light fractions. Expression level of the mRNAs in the heavy fraction and the light fraction was compared relative to the mRNAs’ expression level in unfractionated sample. Results were expressed as mean ± s.d. from three independent experiments. Student t- test was employed to determine if there was statistical significant between Ctrl O/E and eIF4E O/E groups. “*”, “**” and “***” indicate a statistical significance with P < 0.05 and P < 0.001.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Fractionation, Over Expression, Transfection, Expressing

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: a The mRNA expressions of eIF4E, ERα and FOXM1 in MCF-10A, MCF-7, ZR-75, LCC2 and AK-47 cell lines were determined by qPCR. Actin was used as the internal control. Results were expressed as mean ± s.d. from three independent experiments. Linear regression model was employed to determine the correlation. b Protein expressions of eIF4E, ERα and FOXM1 in the indicated cell lines were determined by western blot. Tubulin was used as the loading control. Correlation between the expression levels of ( c ) eIF4E and FOXM1, ( d ) eIF4E and ERα and ( e ) ERα and FOXM in MCF-10A, MCF-7, ZR-75 and LCC2 was determined. AK-47 is an ERα negative cell line, hence correlation were only made from 4 cell lines. Band intensity was measured by ImageJ from three independent experiments. The expression of protein candidates was relative to tubulin. Each of the dots represented one cell line. Linear regression model was employed to determine the correlation. P < 0.05 was regarded as statistical significance.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: a Overexpression of eIF4E did not alter the mRNA stability of ERα and FOXM1. The cells were treated with 5 µg/mL of ActD for 0 hour and 24 h. qPCR was employed to determine the expression of the candidate mRNA. The formula C 24 = C 0 e −kt was employed to determine the decay constant ( k ) of each of the candidate mRNA. C 24 and C 0 were the relative expression of the mRNA at 24-hour and 0-hour. The length of the experiment was t. Overexpression of eIF4E could enhance the transcriptional activity of ERα in ( b ) MCF-7 and ( c ) ZR-75 cells. Luciferase reporter assay was employed. The promoter region contained estrogen receptor response element. The luciferase activity positively correlated with the transcription activity of ERα. Results were expressed as mean ± s.d. from three independent experiments. Student t- test was used to determine statistical significance.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Over Expression, Expressing, Activity Assay, Luciferase, Reporter Assay

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: Overexpression of eIF4E could induce tamoxifen resistance in ( a ) MCF-7 and ( c ) ZR-75 cells. 0.5 μg of pCMV6 was used as control while overexpression of eIF4E was mediated by transfection of 0.5 μg of pCMV6_eIF4E . The effect on eIF4E and FOXM1 expression in ( b ) MCF-7 and ( d ) ZR-75 was confirmed by western blot. Tubulin was used as the loading control. The cells were treated with either ethanol (EtOH) or 4 µM of tamoxifen (TAM). e Knockdown of eIF4E could reverse tamoxifen resistance in LCC2 cells. Knockdown of eIF4E was mediated by transfection of 20 pmol of siRNA#1 or siRNA#2. 20 pmol of non-targeting siRNA (siCtrl) was used. f The knockdown efficiency of the siRNAs on eIF4E and FOXM1 expression in LCC2 was determined by western blot. Tubulin was used as the loading control. The cells were treated with either EtOH or 4 µM of tamoxifen. MTT assay was employed to determine the cell viability. Results were expressed as mean ± s.d. from three independent experiments. “***” indicates both time and the treatment significantly affect the cell viability with a statistical significance with P < 0.001 by two way ANOVA.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Over Expression, Transfection, Expressing, Western Blot, MTT Assay

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: Stable eIF4E overexpressing ( a ) MCF-7 and ( c ) ZR-75 cell lines were used. Knockdown of FOXM1 was mediated by transfection of 50 pmol of the corresponding siRNA#1 or siRNA#2 in the stable eIF4E overexpressing cells. 50 pmol of non-targeting siRNA (siCtrl) was used as control. The cells were treated with either EtOH or 4 µM of tamoxifen. MTT assay was employed to determine the cell viability. Results were expressed as mean ± s.d. from three independent experiments. “***” indicates both time and siRNA treatments significantly affect the cell viability with a statistical significance with P < 0.001 by two way ANOVA. The knockdown effect of FOXM1 by the siRNAs and the expression of eIF4E in ( b ) MCF-7 and (d) ZR-75 was confirmed by western blot. Tubulin was used as the loading control. Knockdown of FOXM1 could resume the effect of tamoxifen on caspase activation in eIF4E overexpressing cells. Knockdown of FOXM1 was mediated by transfection of 50 pmol of the corresponding siRNA#1. Caspase 3/7 activity assay was performed on ( e ) MCF-7 eIF4E O/E and ( f ) ZR-75 eIF4E O/E cells. “***” represents P < 0.001. Student t- test was employed to determine the statistical difference between two groups.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Transfection, MTT Assay, Expressing, Western Blot, Activation Assay, Activity Assay

Journal: Oncogene

Article Title: Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

doi: 10.1038/s41388-020-1210-y

Figure Lengend Snippet: a Representative photomicrographs of high and low expression of eIF4E and ERα in breast tumours in TMA achieve. b Representative photomicrographs of high and low expression of eIF4E and FOXM1 in breast tumours in TMA achieve. IHC was employed to stain the target protein in the 134 cases of ER+ve tumour tissues. c eIF4E scores were correlated with clinically reported ER and PR status obtained from pathology reports. eIF4E was positively correlated with ERα and FOXM1 was observed. Chi-square test was performed to determine the significance. d High expression of eIF4E was associated with tamoxifen resistance. There were 97 breast cancer cases, among them 55 being tamoxifen sensitive and 42 being tamoxifen-resistant. Chi-square test was performed to determine the significance.

Article Snippet: Transient transfections and siRNA knockdown were performed using lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. pCMV6_eIF4E (#SC118908, Origene) was used to overexpress and establish the stable cell lines.

Techniques: Expressing, Staining