Journal: Cell reports
Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation
doi: 10.1016/j.celrep.2023.112646
Figure Lengend Snippet: (A) Representative immunoblots of 3 independent studies of ITG proteins in Nsi and DAP5-silenced MB-231 cells. Dox induction of shRNAs for 4 days followed by immunoblot analysis of equal protein amounts. (B) Representative immunoblots of 3 independent studies of EMT biomarker protein levels in Nsi and DAP5-silenced 4T1 and MB-231 cells carried out as in (A). (C) Representative immunoblots of 3 independent studies of eIF4E and eIF2α proteins in Nsi and DAP5-silenced MB-231 cells carried out as in (A). (D) Representative immunoblots of 3 independent studies to test requirement for eIF3d cap binding activity for DAP5-dependent mRNAs in MB-231 cells. Cells were small interfering RNA (siRNA) silenced for eIF3d for 1 day and transfected with vectors expressing WT, α5, or α11 cap binding mutants of eIF3d for 2 days, and equal protein amounts analyzed using immunoblot. (E) Matrigel Transwell invasion assays performed with 4T1 and MB-231 cells silenced for 24 h with Nsi or DAP5 Dox-inducible shRNAs. Mean number of invading cells/field with SEM from 3 replicates for each cell line. See also . (F) Cell migration wound healing assays performed with 4T1 and MB-231 cells silenced for 24 h with Nsi or DAP5 Dox-inducible shRNAs. Time 0 represents 100% wound separation of the cell layer. Mean of percentage of migrated cell surface areas (percentage closure) with SEM from 3 replicates for each cell line. See also . (G) Induction of apoptosis by cell detachment (anoikis) determined in 4T1 and MB-231 cells silenced for 24 and 48 h for Nsi, DAP5, or eIF4GI, comparing cells maintained on adherent or ultra- low-adherence plates. Percentage cell apoptosis determined by annexin V-fluorescein isothiocyanate (FITC) staining and flow cytometry, quantified using FlowJo software. Mean with SEM of 3 independent studies per cell line. (H) Representative immunoblot of 3 independent studies of DAP5, eIF4G1, eIF3d, and control β-actin protein levels in Nsi or DAP5-silenced 4T1 and MB-231 cells, silenced for 2 days with Dox-inducible shRNAs. Data are represented as mean ± SEM. n.s., not significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired parametric two-tailed t test.
Article Snippet: Rabbit polyclonal anti-human/mouse β-actin antibody , Cell Signaling Technology , Cat# 4967/RRID:AB_330288.
Techniques: Western Blot, Biomarker Discovery, Binding Assay, Activity Assay, Small Interfering RNA, Transfection, Expressing, Migration, Staining, Flow Cytometry, Software, Control, Two Tailed Test
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
