Journal: bioRxiv

Article Title: eIF4E and Ezrin cooperate in pseudopods to drive a localized migratory translation program in acute myeloid leukemia

doi: 10.64898/2026.02.21.707190

Figure Lengend Snippet: A. Western blot of total cell lysates from MM6 cells grown in suspension demonstrated knockdown of Ezrin (siEZR) or eIF4E (siEIF4E) compared to RNAi to luciferase (siLUC) used as a negative control. b-Actin is provided as a loading control. Other proteins of the Ezrin-CD44-HA axis are also shown. Quantification for these is shown in with 3-6 biological replicates for each protein. B. Adhesion and invasion capacity of MM6 cells onto/through HS-5 bone marrow stroma. Fold change relative to siLUC is shown. Each symbol represents a biological replicate performed independently with replicates. Bars represent the mean, shown with standard deviations and p-values (one-way ANOVA). C. Western blot of eIF4E and Ezrin immunoprecipitations (IPs) using total cell lysates from MM6 cells in suspension. SN, supernatant after immunoprecipitation, IgG, negative control. Representative of three biological replicates. IPs of total cell lysates from THP-1 cells in suspension are provided in . D. IPs from MM6 cells in suspension using the rRNA antibody Y10b. LC indicates antibody light chain. Representative of three biological replicates. E. RNA immunoprecipitations (RIPs) from MM6 total cell lysates grown in suspension using anti-Ezrin (Ezrin RIP) or anti-eIF4E (eIF4E RIP) antibodies. Data are from RT-qPCR and represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). F. Western blots of eIF4E and Ezrin IPs from the cytoplasmic fractions of MM6 cells in suspension or G . After invasion through HS-5 bone marrow stroma (invaded). Fractionation controls are provided in . H. RIPs from MM6 cytoplasmic fraction from invaded cells using anti-eIF4E (eIF4E RIP) or anti-Ezrin (Ezrin RIP) antibodies. Data are from RT-qPCR represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). I. Count of the number of pseudopods observed in suspension and invaded MM6 cells represented as a fraction relative to the total cells counted. Each symbol represents a biological replicate. Bars represent the mean, shown with standard deviations and p-values (two-way ANOVA). J . Immunofluorescence and confocal microscopy demonstrating eIF4E, Ezrin, CD44 and rRNA are localized to the same pseudopods (white arrows). All confocal micrographs represent a single section through the plane of the cell. Scale bar = 10 µm

Article Snippet: In case of Y10b IPs, 100 ml of Y10b mouse hybridoma supernatant (DSHB), or 10 mg od mouse IgG (cat# 12-371, Millipore) were bound to the beads 50 μL protein G conjugated superparamagnetic beads in 1 ml of NT2 buffer ON at 4°C, washed three times with NT2 buffer, added to 1 mg of total cell lysates, incubated overnight at 4°C with rotation and washed as described for IPs with rabbit antibodies.

Techniques: Western Blot, Suspension, Knockdown, Luciferase, Negative Control, Control, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Fractionation, Immunofluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: eIF4E and Ezrin cooperate in pseudopods to drive a localized migratory translation program in acute myeloid leukemia

doi: 10.64898/2026.02.21.707190

Figure Lengend Snippet: A. Western blot of total cell lysates from MM6 cells grown in suspension demonstrated knockdown of Ezrin (siEZR) or eIF4E (siEIF4E) compared to RNAi to luciferase (siLUC) used as a negative control. b-Actin is provided as a loading control. Other proteins of the Ezrin-CD44-HA axis are also shown. Quantification for these is shown in with 3-6 biological replicates for each protein. B. Adhesion and invasion capacity of MM6 cells onto/through HS-5 bone marrow stroma. Fold change relative to siLUC is shown. Each symbol represents a biological replicate performed independently with replicates. Bars represent the mean, shown with standard deviations and p-values (one-way ANOVA). C. Western blot of eIF4E and Ezrin immunoprecipitations (IPs) using total cell lysates from MM6 cells in suspension. SN, supernatant after immunoprecipitation, IgG, negative control. Representative of three biological replicates. IPs of total cell lysates from THP-1 cells in suspension are provided in . D. IPs from MM6 cells in suspension using the rRNA antibody Y10b. LC indicates antibody light chain. Representative of three biological replicates. E. RNA immunoprecipitations (RIPs) from MM6 total cell lysates grown in suspension using anti-Ezrin (Ezrin RIP) or anti-eIF4E (eIF4E RIP) antibodies. Data are from RT-qPCR and represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). F. Western blots of eIF4E and Ezrin IPs from the cytoplasmic fractions of MM6 cells in suspension or G . After invasion through HS-5 bone marrow stroma (invaded). Fractionation controls are provided in . H. RIPs from MM6 cytoplasmic fraction from invaded cells using anti-eIF4E (eIF4E RIP) or anti-Ezrin (Ezrin RIP) antibodies. Data are from RT-qPCR represented relative to input. Each symbol represents a biological replicate performed independently with triplicates. Bars represent the mean, shown with standard deviations and p-values (two tailed Welch’s t test). I. Count of the number of pseudopods observed in suspension and invaded MM6 cells represented as a fraction relative to the total cells counted. Each symbol represents a biological replicate. Bars represent the mean, shown with standard deviations and p-values (two-way ANOVA). J . Immunofluorescence and confocal microscopy demonstrating eIF4E, Ezrin, CD44 and rRNA are localized to the same pseudopods (white arrows). All confocal micrographs represent a single section through the plane of the cell. Scale bar = 10 µm

Article Snippet: In case of Y10b IPs, 100 ml of Y10b mouse hybridoma supernatant (DSHB), or 10 mg od mouse IgG (cat# 12-371, Millipore) were bound to the beads 50 μL protein G conjugated superparamagnetic beads in 1 ml of NT2 buffer ON at 4°C, washed three times with NT2 buffer, added to 1 mg of total cell lysates, incubated overnight at 4°C with rotation and washed as described for IPs with rabbit antibodies.

Techniques: Western Blot, Suspension, Knockdown, Luciferase, Negative Control, Control, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Fractionation, Immunofluorescence, Confocal Microscopy

Journal: Aging Cell

Article Title: Translational Remodeling of the Synaptic Proteome During Aging

doi: 10.1111/acel.70262

Figure Lengend Snippet: Validation of ribosomal transcripts and proteins regulation in synaptosomes during aging. (A) Western blot of ribosomal proteins using total cortical homogenate and synaptosomes of young, adult and old animals. Tubulin was used as loading control in all the experiments. (B) Left panel: Mean of protein intensity quantification in Total Homogenate (TH) relative to tubulin for each time point of panel (A). Right panel: Mean of protein intensity quantification in synaptosomes (SYN) relative to tubulin for each time point of panel (A). (C) Left panel: Mean of ribosomal transcripts expression in total homogenate (TH) relative to Ldhb for each time point using independent samples. Right panel: Mean of ribosomal transcripts expression in synaptosomes (SYN) relative to Ldhb for each time point using independent samples. Each time point N = 4. (D) Ribosomal RNA (rRNA) in total homogenate (TH) and synaptosomes (SYN). Box plot of the fold change of 18S and 28S between SYN and TH across the ages. Red indicates young animals ( N = 4), blue adult animals ( N = 4) and green old animals ( N = 4). (E) Targeted proteomics of ribosomal proteins in SYN. Normalized ratios of light to heavy peptides displayed. p values estimated by one‐way ANOVA. (F) Immunofluorescence of SYN isolated from young, adult and old animals ( N = 4 for each time point). Synaptic protein synaptophysin (SYP) stained in green, in red Ribosomal protein RPL7 and in blue Ribosomal RNA. Bar 5 μm. Right panel: Quantification of mean fluorescence intensity of RPL7 using SYP as reference. Each dot represents a single synaptosome, bars indicate the mean with 95% Confidence Interval (CI). (F) Isolated synaptosomes staining. Synaptosomes were immunostained with synaptophysin (SYP) (green), RPL7 (red), and Y10b (blue). On the right the mean intensity of RPL7 channel compared to SYP channel in the three time points is reported ( p < 0.0001 evaluated by Kruskal–Wallis test).

Article Snippet: The primary antibodies used were the following: Synaptophysin (Synaptic Systems, Cat. No. 101004), RPL7 (Bethyl, Cat. No. A300‐741A), and rRNA (Y10b) (Novus biologicals, Cat. No. NB100‐662).

Techniques: Biomarker Discovery, Western Blot, Control, Expressing, Targeted Proteomics, Immunofluorescence, Isolation, Staining, Fluorescence

Journal: Nature

Article Title: Probing condensate microenvironments with a micropeptide killswitch

doi: 10.1038/s41586-025-09141-5

Figure Lengend Snippet: a , Schematic of the NuFANCI procedure. b , Protein expression profiles of NuFANCI-isolated nucleoli and their respective input samples. In the left four columns, nucleolar proteins are annotated based on previous proteomics data. N, two clusters of nucleolar proteins. LFQ, label-free quantification. c , The proteomes of GFP-nb–KS-targeted nucleoli and GFP-nb–KS F-to-G -targeted nucleoli compared with the anti-GFP-nanobody control. P values were calculated using one-sided Student’s t -tests. FC, fold change. d , Fixed-cell immunofluorescence images of GFP–NPM1-expressing cells that were untransfected or transfected with GFP-nb, GFP-nb–KS, GFP-nb–2×KS or GFP-nb–KS F-to-G constructs. Scale bars, 5 µm. e , NEPRO fluorescence intensity in nucleoli. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Dunnett’s T3 multiple-comparison test versus the GFP-nb condition. n = 32 (untransfected), 33 (GFP-nb), 36 (GFP-nb–KS), 29 (GFP-nb–2×KS) and 31 (GFP-nb–KS F-to-G ) cells from two independent experiments. f , NEPRO fluorescence intensity in nuclei. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Dunnett’s T3 multiple-comparison test versus the GFP-nb condition. The numbers of cells are the same as described in e and are from two independent experiments. g , FRAP analysis of mCherry-signal in HCT-116 cells expressing GFP–NPM1 from the endogenous locus after transfection with the indicated nb constructs and co-transfection with mCherry–SURF6 or mCherry–RPL18. Data are mean ± s.d. n = 10 for all, except for the SURF6 experiment for the TagBFP-only sample ( n = 8; asterisk). h , Fixed-cell immunofluorescence images of 5.8S rRNA in GFP–NPM1-expressing cells that were transfected with either the GFP-nb or GFP-nb–2×KS construct. Scale bars, 5 µm. i , Quantification of 5.8S rRNA mean fluorescence intensities in the demixed and remaining portions of nucleoli in GFP-nb–2×KS-expressing cells. Data are mean ± s.d. n represents the number of cells from one experiment. The experiment was repeated twice with similar results. P values were calculated using one-way ANOVA followed by Tukey’s post hoc test. Some elements in the scheamtic in a were created in BioRender. Hnisz, D. (2025) https://BioRender.com/fa5zmne .

Article Snippet: The following primary antibodies were used: 5.8S rRNA (Novus, NB100-662SS, 1:500), HA-tag (Cell Signaling, C29F4, 1:1,000), NEPRO (Santa Cruz, sc-376579, 1:100), RNAPII (Abcam, ab26721, 1:500) and H3K27Ac (Abcam, ab4729, 1:1,000).

Techniques: Expressing, Isolation, Quantitative Proteomics, Control, Immunofluorescence, Transfection, Construct, Fluorescence, Comparison, Cotransfection