PMC

Depletion of eIF4G1 sensitizes cells to IR-mediated autophagy, apoptosis, and senescence. (A) Equal amounts of protein lysates from MCF10A cells stably silenced for eIF4G1 or NS, treated with 8 Gy IR, resolved by SDS/PAGE, and immunoblotted with antibodies as shown. LC3-II is a marker for induction of autophagy. (B) FACS analysis conducted on 8 Gy–irradiated NS or eIF4G1-silenced MCF10A cells stained with PE-Annexin V, a marker of apoptosis. (C) Cells stably transfected with NS or eIF4G1 shRNA lentiviruses were subjected to 0 or 4 Gy irradiation, and β-gal assays for cellular senescence were performed. Representative phase-contrast photomicrographs of cells of triplicate studies shown at 100× magnification. Arrows mark β-gal staining. Assays were n = 3.

Effect of eIF4G1, eIF4G2 and eIF4E silencing on overall protein synthesis and cell survival in IR-treated cells. (A) NS (non-silencing) and eIF4G1-silenced MCF10A cells were treated with 8 Gy IR or mock-treated. Protein synthesis rates determined by [³⁵S]methionine incorporation, normalized to NS-shRNA nonirradiated control (0 h). SEMs are shown. Immunoblot is eIF4G1 in silenced and control cells. (B) Equal amounts of protein at 24 h post-IR were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. (C) Clonogenic survival assays performed on MCF10A cells following IR, with stable shRNA to eIF4E, eIF4G1, eIF4G2, or an NS random sequence. Cells were plated overnight and irradiated, and colonies were scored after 2 wk (n = 3). (D) Immunoblot of equal protein amounts of lysates from C.

DNA-damage response signaling and G₂/M arrest are impaired in eIF4G1-silenced cells. (A) Kinetics of IRIF in MCF10A cells stably expressing NS or eIF4G1 shRNAs assessed by confocal microscopy. Actin: direct immunofluorescence with FITC-phalloidin; γH2AX (H2AX-S139P): primary monoclonal antibody and TRITC anti-mouse secondary antibody; 53BP1: rabbit primary antibody coupled to Alexa Fluor 488 anti-rabbit secondary antibody (n = 3). (B) Equal protein amounts of total cell lysates analyzed by SDS/PAGE and immunoblot with antibodies for protein and phospho-specific proteins as shown. (C) Cell cycle analysis performed by propidium iodide staining of RNase A treated MCF10A NS and eIF4G1-silenced cells following IR. DNA content (x axis) was determined by FACS analysis.

eIF4E requirement of selectively translated DDR and survival mRNAs with eIF4G1 overexpression. (A) Cells silenced with NS or eIF4G1 shRNAs subjected to 8 Gy IR and fractionated by sucrose density gradient sedimentation into unbound mRNA, light (1–3) and heavy (≥4) ribosomes. (B) Immunoblot of NS and eIF4G1-silenced MCF10A cells at 12 h following 8 Gy IR from equal protein amounts of total cell lysates. (C) Relative rate of protein synthesis assessed by immunoprecipitation (IP) of de novo proteins shown following 2 h metabolic labeling with [³⁵S]methionine with actinomycin D (20 µM) and MG132 (10 µM) at 2 h after 8 Gy IR. (D) Immunoblot of NS, eIF4E silenced or eIF4G1-silenced MCF10A cells following 8 Gy IR at 24 h, from equal protein amounts of total cell lysates. (E) Quantification of immunoblot studies shown in B. EMCV results derived from Renilla luciferase reporter studies (n = 3).