rabbit monoclonal anti eif6  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: RNF10 and RIOK3 facilitate 40S ribosomal subunit degradation upon 60S biogenesis disruption or amino acid starvation

doi: 10.1016/j.celrep.2025.115371

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-eIF6 , Cell Signaling Technology , Cat# 3833; RRID: AB_2096520.

Techniques: Recombinant, Multiplex sample analysis, Protease Inhibitor, Western Blot, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Viability Assay, Luciferase, Knockdown, Plasmid Preparation, Software

Journal: Experimental gerontology

Article Title: Mechanistic insights into EIF6 as a target of Apigenin in alleviating chondrocyte senescence.

doi: 10.1016/j.exger.2025.112725

Figure Lengend Snippet: Figure2. The EIF6 locus for KOA. (A) Top plot, P values from GWAS for KOA (brown dots) and P value from the SMR test (diamond) using the Westra eQTL data. Bottom plot, P values from the eQTL studies for EIF6. Shown are all the SNPs available in the GWAS and eQTL data. (B) Effect sizes of SNPs from GWAS plotted against those for SNPs from the EIF6. The orange dashed lines represent the estimate at the top cis-eQTL. Error bars are the standard errors of SNP effect. (C) Heat map of multi-tissue colocalization heatmap. KOA, Knee osteoarthritis. GWAS, Genome-wide association studies. SMR, Summary-data-based Mendelian Randomization.

Article Snippet: The proteins were transferred to membranes, then incubated with a EIF6 antibody (Manufacturer: Proteintech, China) for a duration of 4 °C overnight.

Techniques: GWAS

Journal: Experimental gerontology

Article Title: Mechanistic insights into EIF6 as a target of Apigenin in alleviating chondrocyte senescence.

doi: 10.1016/j.exger.2025.112725

Figure Lengend Snippet: Figure3. Three-dimensional schematic of molecular docking between Apigenin and EIF6. On the left, the spherical structure is depicted, while on the right, the rod- shaped structure is shown.

Article Snippet: The proteins were transferred to membranes, then incubated with a EIF6 antibody (Manufacturer: Proteintech, China) for a duration of 4 °C overnight.

Techniques:

Journal: Experimental gerontology

Article Title: Mechanistic insights into EIF6 as a target of Apigenin in alleviating chondrocyte senescence.

doi: 10.1016/j.exger.2025.112725

Figure Lengend Snippet: Figure4. EIF6 may be associated with aging pathways via single-cell sequencing. (A) A t-SNE diagram demonstrates the APEC clustering of chondrocytes from cartilage, each cluster represents different cell clusters. (B) t-SNE cluster maps show clustering grouped by low and high EIF6 expression. (C) A bubble chart depicts the enrichment

Article Snippet: The proteins were transferred to membranes, then incubated with a EIF6 antibody (Manufacturer: Proteintech, China) for a duration of 4 °C overnight.

Techniques: Sequencing, Expressing

Journal: Experimental gerontology

Article Title: Mechanistic insights into EIF6 as a target of Apigenin in alleviating chondrocyte senescence.

doi: 10.1016/j.exger.2025.112725

Figure Lengend Snippet: Figure5. Apigenin suppresses chondrocyte senescence by upregulating EIF6 expression. (A) Cell viability of cells treated with hydrogen peroxide (H2O2) was analyzed using a CCK8 assay (n = 3). (B) Cell viability of cells treated with Apigenin was analyzed using a CCK8 assay (n = 3). (C) Cell viability of cells treated with Apigenin (20 μM) or H2O2 (350 μM) was analyzed using a CCK8 assay (n = 3). (D) Senescence was determined by SA-β-Gal staining (blue) (n = 3). (E) Western blot of

Article Snippet: The proteins were transferred to membranes, then incubated with a EIF6 antibody (Manufacturer: Proteintech, China) for a duration of 4 °C overnight.

Techniques: Expressing, CCK-8 Assay, Staining, Western Blot

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Disruption of eIF6 and uL14 interactions through N106S mutation induces p53 checkpoint response. ( A ) Cryo-electron microscopy (EM) structure of human eIF6 interacting with uL14 (RPL23) of pre-60S ribosomal subunit (PDB: 6LU8). The key residues of interaction in eIF6 (N106) and the terminal 8 residues (8AA) in uL14 are highlighted. ( B ) Representative western blot of HCT116 cells with homozygous knock-in of eIF6-N106S mutation in comparison to isogenic WT controls. Blots probed with anti-eIF6 antibody (Santa Cruz Biotechnology) and anti-β-tubulin antibody (loading control). ( C ) Representative western blots were probed with anti-p53, anti-p21 and anti-γ H2AX antibodies. β-Tubulin, β-catenin and β-actin are loading controls. (D and E) Blots represented in panel (C) were quantitated and normalized to loading control. Plots represent standard error of the mean of four ( D ) and three ( E ) independent experiments. Significant differences in p53 ( P = 0.0039) (panel D) and p21 levels ( P = 0.000 047) (panel E) relative to WT were determined by an unpaired two-tailed t -test.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Disruption, Mutagenesis, Cryo-Electron Microscopy, Western Blot, Knock-In, Comparison, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Increased vacant 80S levels and reduced rate of global translation due to weakened binding of eIF6-N106S mutant to the 60S subunit. ( A ) Representative polysome profiles of eIF6-N106S relative to eIF6-WT assayed by sucrose gradient centrifugation with a gradient of 20–47% sucrose. Arrows indicate differences in 80S monosomal peak (solid arrow) and polysome peak (dashed arrow). ( B ) Representative western blots of proteins extracted from the indicated sucrose-gradient fractions. Blots represent three independent experiments. Blots were probed with anti-eIF6 and anti-uL14 antibodies. ( C ) Western blot represents eIF6 and β-tubulin (loading control) levels in the input for polysome profile assay. ( D ) Plot shows the relative AUC measurements for 80S monosome levels in eIF6-WT and eIF6-N106S mutant. Plot shows standard error of the mean of four independent experiments with a significant difference between eIF6-WT and eIF6-N106S ( P = 0.0000 000 003) determined by an unpaired two-tailed t -test. ( E ) Western blot represents SERBP1 and uL14 levels in fractions 4, 5 and 6 of polysome profiles (three independent replicates). ( F ) Plot shows normalized AHA incorporation rates assayed in triplicate per experiment and depicted as standard error of the mean of three independent experiments. Significant difference between eIF6-WT and eIF6-N106S ( P = 0.00 001) determined using an unpaired two-tailed t -test.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Binding Assay, Mutagenesis, Gradient Centrifugation, Western Blot, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Nucleolar localization of eIF6 is not dependent on its interaction with BCCIP or uL14. ( A ) Western blot represents the nuclear (nuc) and cytoplasmic (cyto) distribution of eIF6. Blots were probed with anti-eIF6 (Santa Cruz Biotechnology), anti-topoisomerase II-β (nuclear marker) and anti-β-tubulin (cytoplasmic marker) antibodies. Adjacent western blots represent the total input levels of eIF6 and β-tubulin (loading control). (B and C) Blots shown in panel (A) were quantitated and the percent distribution of eIF6 in nuclear and cytoplasmic fractions were plotted for eIF6-WT ( B ) and eIF6-N106S mutant ( C ). Plots indicate the standard error of the mean of three independent experiments. ( D ) IF images of asynchronous cells show localization of eIF6 to nucleoli (arrows) in both WT and mutant cells and decreased nuclear localization in mutant. Fixed cells were stained with DAPI (nuclear marker) or with anti-eIF6 antibody. Scale bar highlighted in the top left image represents scale bars for all images shown in panel. ( E ) Plot indicates standard error of the mean of eIF6 fluorescence intensity in nucleus normalized to DAPI intensity per cell. Significant difference ( P = 0.03) determined using an unpaired two-tailed t -test. ( F ) Representative western blot shows IP of endogenous eIF6 or anti-mouse IgG control and its interaction with endogenous BCCIP. The corresponding input is shown. Vinculin used as loading control. ( G ) Representative western blot shows IP of endogenous BCCIPβ or anti-rabbit IgG control and its interaction with endogenous eIF6. The corresponding input is shown. Vinculin used as loading control. (*Heavy-chain IgG migrates close to BCCIPβ).

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Western Blot, Marker, Control, Mutagenesis, Staining, Fluorescence, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Deregulation of mitotic translation but unaltered regulation of other eIFs in eIF6-N106S mutant. ( A–D ) Representative western blots of eIF6-WT and eIF6-N106S probed with the indicated antibodies. Anti-vinculin, anti-β-tubulin or anti-GAPDH antibodies were used as loading controls. Serine-51 phosphorylation of eIF2α was induced by oxidative stress with 4mM H 2 O 2 for 1 h. ( E ) Global translation levels in asynchronous cells (Async) or mitotic cells synchronized with nocodazole measured using OPP incorporation assay. Plots represent the standard error of the mean of OPP incorporation of replicates from three independent experiments normalized to eIF6-WT asynchronous rates. Significant differences between WT-async and WT-mitosis ( P = 0.000 000 002), WT-async and N106S-async ( P = 0.000 000 021) and WT-async and N106S-mitosis ( P = 0.000 000 003), and no-significant (ns) differences between N106S-async and N106S-mitosis ( P = 0.97) and WT-mitosis and N106S-mitosis ( P = 0.91) as determined by a one-way ANOVA and Sidak’s multiple comparisons test. ( F ) Plot shows cell cycle profiles determined by flow cytometry. Data depict the standard error of the mean of three independent experiments. Significant differences between eIF6-WT and eIF6-N106S for G1 phase ( P = 0.017) and G2/M phase ( P = 0.0105) of cell cycle as determined by an unpaired two-tailed t -test.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Mutagenesis, Western Blot, Phospho-proteomics, Flow Cytometry, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: eIF6-N106S mutation leads to errors in chromosome segregation, mitotic exit delay and mitotic catastrophe. ( A and B ) Representative IF images of binucleated cells in mutant. Fixed asynchronous cells stained with DAPI (nuclear stain) or anti-tubulin antibody. Scale bar highlighted in the first image represents scale bars for all images in panel. Panel (B) shows the standard error of the mean of binucleated cells from three independent experiments. A total of ∼900 cells counted per genotype for three experiments combined. Statistical significance ( P = 0.0106) determined using an unpaired two tailed t -test. ( C and D ) Representative IF images show mitotic catastrophe with multinuclei, giant nuclei, micronuclei and vacuoles (white arrow). Panel (D) shows the standard error of the mean of giant and multinucleated cells from three independent experiments. Statistical significance ( P = 0.0002) determined using an unpaired two tailed t -test. ( E and F ) Representative IF image shows binucleate cell with nuclei tethered by chromatin bridge (white arrow). Panel (F) shows the standard error of the mean of nuclei tethered by chromatin bridges during cytokinesis from a total of ∼400 asynchronous cells from three independent experiments and P = 0.0017 as determined by an unpaired two-tailed t -test. ( G and H ) Representative IF images show anaphase bridges in mitotic cells. Panel (H) indicates standard error of the mean of mitotic cells exhibiting segregation defects (anaphase bridges and/or lagging chromosomes) from three independent experiments. Statistical significance ( P = 0.0014) determined using an unpaired two tailed t -test. ( I and J ) Confocal time-lapse brightfield images show snapshots of cells (white arrows) progressing through metaphase to early G1. Cells synchronized in G2 phase with RO-3306 for 20 h and released into mitosis for imaging. Timing of progression from metaphase to G1-entry plotted (panel J). A total of 25 (eIF6-N106S) and 30 (eIF6-WT) cells undergoing mitosis were imaged. Statistical significance ( P = 0.0005) determined using an unpaired two tailed t -test.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Mutagenesis, Staining, Two Tailed Test, Imaging

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Ribosome profile analysis shows altered TEs of transcripts associated with mitotic kinetochore organization, chromosome localization and cytoskeleton in eIF6-N106S mutant. ( A ) Scatter plot shows the distribution of transcripts with a significant downregulation or upregulation in TEs of 1.5-fold or more in mutant compared with WT (ΔTE) and non-significant ΔTEs. ΔTE calculations are indicated in supplementary material. (B and C) Box plot shows changes in levels of mRNA ( B ) and RPFs ( C ) for transcripts with a significant 1.5-fold or more upregulation (green boxes) or downregulation (red boxes) in TEs in mutant compared with WT. Plots also depict non-significant ΔTEs (gray boxes). ANOVA test shows significant differences among the four classes ( P < 2e−16) and significant differences between each group ( P < 2e−16) according to Tukey’s Honestly Significant Difference (HSD) post hoc test. (D and E) GO/KEGG-enrichment analysis was carried out on transcripts that were downregulated ( D ) or upregulated ( E ) by 1.5-fold or more in the mutant compared with WT. Moreover, P -values were determined using Fisher’s one-tailed t -test performed by g:profiler. Read lengths for ribosome profiling and sample size are indicated in the supplementary material.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Mutagenesis, One-tailed Test

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: eIF6-N106S mutation inhibits colonic cancer invasion in a subcutaneous tumor xenograft model. ( A ) Bioluminescence image shows stable transfectant clones of eIF6-WT and eIF6-N106S expressing similar firefly luciferase activity. ( B ) Luciferase activity for images shown in panel (A) quantitated and plotted as average radiance. Plot indicates standard error of the mean of three independent experiments. Difference between eIF6-WT and eIF6-N106S was not significant ( P = 0.94) as determined by an unpaired two-tailed t -test. ( C ) Plot indicates FC in cell proliferation rates relative to 0 h of plating as measured by MTS assay. Plot indicates standard error of the mean of three independent experiments with triplicate wells measured per experiment. The difference between eIF6-WT and eIF6-N106S was significant at 24 h ( P = 0.000 001), 48 h ( P = 0.000 001) and 72 h ( P = 0.0005) of growth as determined by an unpaired two-tailed t -test. ( D ) Plot indicates the average luciferase activity in mice injected with eIF6-WT ( n = 8 mice) and eIF6-N106S cells ( n = 8) on the day of injection (Day 0). The difference between eIF6-WT and eIF6-N106S was not significant ( P = 0.98) as determined by an unpaired two-tailed t -test. ( E ) Representative images show bioluminescence signal measured from Day 0 to Week 4 before mice were sacrificed. Photon flux is indicated by pseudo-colored heatmap and tumor-specific luciferase activity was measured by defining ROI. The presence of luciferase activity in other parts of the body beyond the subcutaneous injection site indicated with arrows. ( F ) Representative ventral view of mice showing bioluminescence signal at Week 4. ( G ) Table indicates number of mice exhibiting invasive cancer relative to total number of mice.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Mutagenesis, Transfection, Clone Assay, Expressing, Luciferase, Activity Assay, Two Tailed Test, MTS Assay, Injection

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Overexpression of eIF6 observed primarily in high-grade invasive bladder and breast cancers. ( A and B ) Western blot probed for eIF6 in human bladder cancer cell lines and healthy (normal) bladder epithelial cells. β-Tubulin used as loading control. Blots shown in panel (A) were quantitated and eIF6 levels were normalized to loading control and plotted (panel B) as FC over eIF6 levels in normal bladder epithelial cells. Values indicate standard error of the mean from four independent experiments and significant differences for T24 ( P = 0.0065), UMUC3 ( P = 0.0132) and HT1197 ( P = 0.0017) determined by an unpaired two-tailed t -test. ( C ) Images represent eIF6 expression in patient-derived benign and high-grade tumors by immunohistochemistry using anti-eIF6 antibody. Enlarged inset shows the presence of eIF6 in nucleoli and cytoplasm in high-grade cancers. ( D ) Images shown in panel (C) were quantitated and eIF6 expression in patient-matched benign tissues relative to high-grade cancers (six patients) were plotted. Significant differences were determined using an unpaired two-tailed t -test ( P = 0.0063). ( E ) Plot shows eIF6 expression in unmatched benign tissues, high-grade and low-grade bladder cancers derived from patients. ( F and G ) Western blot represents high levels of eIF6 in high-grade invasive human triple negative breast cancer cell line. β-Tubulin used as loading control. Blots shown in panel (F) were quantitated and eIF6 levels were normalized to loading control and plotted (G) as FC over eIF6 levels in normal (healthy) HME-1 cells. Values indicate standard error of the mean from four independent experiments and significant differences for MDA-MB-231 ( P = 0.001) determined by an unpaired two-tailed t -test.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Over Expression, Western Blot, Control, Two Tailed Test, Expressing, Derivative Assay, Immunohistochemistry

Journal: Nucleic Acids Research

Article Title: Sequestration of ribosomal subunits as inactive 80S by targeting eIF6 limits mitotic exit and cancer progression

doi: 10.1093/nar/gkae1272

Figure Lengend Snippet: Model depicts the effect of disrupting eIF6 interaction with the 60S subunit. eIF6-N106S mutation weakens its interaction with the 60S subunit. This leads to an increase in vacant 80S ribosomes clamped by SERBP1. However, it does not affect levels or activity of other key eIFs, suggesting that the increase in vacant 80S is likely due to spurious interactions between 40S and 60S in the absence of eIF6 inhibiting subunit joining. In addition, 60S biogenesis is not affected. However, only a fraction of 80S ribosomes is inactive and this could be limited by the availability of free 40S subunits and due to clamping of only a fraction of vacant 80S ribosomes. The increase in vacant 80S ribosomes contributes to global translation repression and deregulation of mitotic translation that causes mitotic exit delays and mitotic catastrophe. The TEs of transcripts with long 3′ UTRs and some mitotic factors and cytoskeletal factors are decreased. It also leads to chromatin and cytoskeletal rewiring that inhibits cancer invasive properties.

Article Snippet: The following antibodies were diluted in TBS-T buffer: eIF6 (1:1000; Santa Cruz Biotechnology), eIF6 (1:1000; Cell Signaling), RPL23 (1:2500; Bethyl), β-tubulin (1:2500; Cell Signaling), GAPDH (1:6000; Cell Signaling), vinculin (1:1000; Cell Signaling), β-actin (1:10 000; Cell Signaling), p53 (1:1000; Santa Cruz Biotechnology), p21 (1:1000; Santa Cruz Biotechnology), BCCIP (1:1500; Bethyl), eIF4E (1:1000; Cell Signaling), 4EBP1 (1:1000; Cell Signaling), 4EBP2 (1:1000; Cell Signaling), eIF2α (1:1000; Cell Signaling), eIF2A (1:1000; Bethyl), eIF4G1 (1:1000; Cell Signaling), topoisomerase IIβ (1:1000; BD Biosciences), eIF4G2 (1:4000; Bethyl), Shwachman-Bodian-Diamond syndrome factor (SBDS) (1:1000; Cell Signaling), vasodilator-stimulated phosphoprotein (VASP) (1:1000; Cell Signaling), Cdc42 (1:1000; Cell Signaling), β-catenin (1:1000; Santa Cruz Biotechnology), E-cadherin (1:1000; Santa Cruz Biotechnology), nuclear factor I/B (NFIB) (1:2000 in 2% milk; Bethyl), Snail (1:1000; Cell Signaling), SERBP1 (1:3500; Bethyl) and PABP1 (1:1000; Cell Signaling).

Techniques: Mutagenesis, Activity Assay