Fig 2: Cardiac glycosides inhibit eukaryotic translation initiation factor 4A complex (eIF4A)-dependent translation in triple-negative breast cancer (TNBC) cells. (A). (CGG)4 Luc2-tdTomato readings in MDA-MD-231-S, MDA-MB-Bone-un, and SUM-159PT cells following treatment with 0-187.5 nM digoxin (12.5 nM increments). (B). Calculated (CGG)4 IC50 values for each cell line. (C). (CGG)4 Luc2-tdTomato/total protein readings (A595) in MDA-MD-231-S cells following treatment with either 0-150 nM digoxin or rocaglamide A. (D). CMV-Luciferase/RTK Renilla expression readings in 293 cells following treatment with 0–100 nM digoxin (* indicates p < 0.05 as analyzed by a one-way ANOVA with a Bonferroni post hoc test). (E). Western blots following treatment with 50 nM rocaglamide A for 48 h in SUM-159PT cells. Band intensities were calculated by densitometry based on the ratio of each target gene (BIRC5 or ROCK1) normalized to the β-actin protein signal with 0 nM set to 100. (F). Western blots following treatment with 0–50 nM digoxin for 48 h in SUM-159PT cells. Band intensities were calculated based on the ratio of each target gene (BIRC5 or ROCK1) normalized to the β-tubulin protein signal with 0 nM set to 100. (G). Western blots following treatment with 50 nM of each indicated drug for 72 h in SUM-159PT cells. Band intensities were calculated based on the ratio of each target gene (eIF4A, eIF4G, or eIF4E) normalized to the β-tubulin protein signal with 0 nM set to 100. Uncropped Blots are shown in Figure S2. (H). Quantitation of mRNA levels by qPCR from SUM-159PT cells following the treatment of 50 nM digoxin. Data were analyzed using the ΔΔCt method with β-tubulin as the control. Relative expression was calculated with DMSO-treated cells set to 100% (* indicates p < 0.05 as analyzed by a one-way ANOVA with a Bonferroni post hoc test).

Fig 3: Western blot analysis showing depletion of eIF4A-1 protein in LdPfn+/-cells, compared to LdPfn+/+ and LdPfn+/-comp cells.(A)(a) Coomassie blue-stained 12% SDS-polyacrylamide gel electrophoretogram showing equal loading of total cell lysates of LdPfn+/+, LdPfn+/-comp and LdPfn+/- cells. Mr, molecular weight markers; lane 1, LdPfn+/+ cell lysate; lane 2, LdPfn+/-comp cell lysate; lane 3, LdPfn+/- cell lysate. (b) Western blot of ‘a’ using anti-LdPfn antibodies. Mr, molecular weight markers; lane 1, LdPfn+/+ cells lysate showing expression of native profilin; lane 2, LdPfn+/-comp cells lysate showing expression of both episomally expressed GFP-LdPfn (43kDa) and native LdPfn (16kDa); lane 3, LdPfn+/- cells lysate showing depletion in the expression of native profilin. (c) Western blot of ‘a’ using anti-β-tubulin antibodies as loading control. Mr, molecular weight markers; lane 1, LdPfn+/+ cells lysate; lane 2, LdPfn+/-comp cells lysate; lane 3, LdPfn+/-cells lysate. For generating LdPfn+/-comp cells, the positive clone of GFP-LdPfn was transfected into LdPfn+/- cells, as described earlier [11]. Hence, there are two bands, one for native LdPfn (16KDa) and other for GFP-LdPfn (43KDa), for profilin in LdPfn+/-comp cells lysate immunoblot. (d) Western blot of ‘a’ using anti-eIF4A-1 antibodies. Mr, molecular weight markers; lane 1, LdPfn+/+ cells lysate; lane 2, LdPfn+/-comp cells lysate; lane 3, LdPfn+/- cells lysate, showing depletion in eIF4A-1 expression levels in LdPfn +/-cells, compared to LdPfn+/+ and LdPfn+/-comp cells. (B) The western blots of three independent experiments have been quantified using GelQuant.net software and the fold change in the protein expression levels of eIF4A1 was calculated by normalizing them against bands of β-tubulin. Around 50% reduction in protein expression levels of eIF4A1 was observed in LdPfn+/- cells, compared to LdPfn+/+ and LdPfn+/-comp cells.

Fig 4: Translation initiation factors localize at the centrosome in HEK293 cells. (a) Immunofluorescence experiments demonstrate that eIF4G and eIF4E colocalize with γ, a centrosomal marker (arrows). (b) Silencing of eIF4E results in downregulation of the signal, confirming the specificity of the immunostaining of the eIF4E antibody. (c) Single channel IF are presented for each protein analysed, arrowheads indicate the staining that suggest the centrosomal localization. (d) Magnification of IF experiments showing colocalization of eIF3B, eIF3G, eIF4E, eIF4A1, eIF4G with centrosomal markers (OFD1 or γtubulin). (e) Analysis of centrosomal localization of eIF4E and eIF3B by Superresolution microscopy. IF (left panels) and 3D reconstruction (right panels) are provided. Bar = 10 μm. For Superresolution images Bar = 2 μm. For all panels n = 3.

Fig 5: BSH-11R bound to the translation-related proteins. (A) BSH interacted with poly(A)-biding protein 1 (PABP1) and polyR allowed BSH to bind with the translation initiation factors and termination factor. (B) The interaction between PABP1 and BSH was RNA-dependent. The BSH-polyR interactions of eIF4A, eIF4G, and eRF3 appear dependent on the polyR part. (C) A Duolink assay confirmed the in situ interactions between BSH-11R and PABP1 or eIF4G in U251MG cells.