EMBO/SourceData huggingface dataset:
Figure 1C. Western blot (WB) analysis of splicing factors for Vector and 2FLAG-eIF4E U2OS cell lines. Myc, Mcl1, and cyclin D1 (CCND1) served as positive controls, and β-actin as a loading control. Both 2FLAG-eIF4E (2Flag-eIF4E) and endogenous eIF4E (endog-eIF4E) are shown. Each β-actin blot corresponds to the above western blots. Experiments were carried out at least three independent times, and one representative experiment is shown.D. WB analysis of splicing factors as a function of eIF4E reduction using CRISPR-eIF4E and CRISPR-Ctrl U2OS cell lines. Myc, Mcl1 and CCND1 served as positive controls, and β-actin a loading control. Each β-actin blot corresponds to the western blots immediately above. Experiments were carried out at least three independent times, and one representative experiment is shown.H. Total RNA levels monitored by RT-qPCR corresponding to mRNA export assays shown in G. Data were normalized to Vector control to calculate fold change. The mean and standard deviation, , as well as p-values (using Student t-test), were derived from three biological experiments, each carried out in technical triplicate. Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001
Figure 2A. WB analysis of the impacts of S53A 2FLAG-eIF4E (2Flag-S53A) relative to WT 2FLAG-eIF4E (2Flag-eIF4E) on SF production. CCND1 served as positive controls, and β-actin or HSP90 as loading controls. Both 2FLAG-eIF4E (2Flag-eIF4E) and endogenous eIF4E (endog-eIF4E) are shown. Each β-actin or HSP90 blot corresponds to the above western blots. S53A eIF4E protein levels are indistinguishable from eIF4E. Experiments were carried out using three separate clones for eIF4E, S53A and Vector controls, and one representative experiment is shown. (Right) Western blots were quantified by FIJI, intensities normalized to Vector control and plotted using PRISM. The mean, standard deviation, and p-values are shown. Student t-test: * p<0.05, **p<0.01, ***p<0.001.B. Polysomal loading analysis was measured to assess the relevance of eIF4E's capacity to increase translation efficiency on SF protein production. Top panel shows the polysomal profile (at 254 nm) demonstrating that 2FLAG-eIF4E overexpression did not alter the profile relative to Vector controls, consistent with previous studies Polysomes were isolated by size exclusion chromatography (SEC) and thus the heaviest polysomes elute first and monosomes last. (Middle and Bottom panels) RNAs were monitored on heavy, light or monosome polysomes using RT-qPCR and presented as a fraction for the given RNA. Known translation targets of eIF4E, MYC and VEGF was shifted to higher polysomes in eIF4E overexpressing cells as well as SF-encoding RNAs with altered polysomes (middle) or those that were unchanged by eIF4E including the negative control ACTB are in the bottom panel Experiments were carried out in biological duplicates using different clones. Means are shown and p-values calculated using ANOVA (PRISM) . Data information: ANOVA test, * p<0.05, **p<0.01, ***p<0.001
Figure 3B. Western blot analysis of untreated (Ctrl) and Ribavirin treated (Rib) Vector and eIF4E NOMO-1 cell lines. Myc, and Mcl1 served as positive controls, and β-actin as a loading control. Each β-actin blot corresponds to the above WBs. Experiments were carried out at least three independent times, and one representative experiment is shown. Ribavirin dose was 10uM which is clinically achievableC. WB analysis of Splicing Factors levels in primary AML samples with high (AML-H) or normal (AML-N) eIF4E levels, as well as bone marrow mononuclear cells from healthy volunteers (Norm for normal). Numbers refer to different individuals. β-actin was used as a loading control. (* = degraded sample).E. Total levels of UsnRNA in NOMO-1 Vector and eIF4E cells monitored by RT-qPCR. Data were normalized to Vector control and shown as a fold change. The mean and standard deviation, as well as p-values (using Student t-test) were derived from three biological replicates each carried out in triplicate for all panels.
Figure 4A. Endogenous eIF4E co-immunoprecipitated with SFs in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates and anti-eIF4E antibody (eIF4E-IP) or appropriate IgG control (IgG-IP). IP samples along with Input (2%) and supernatants (Sn) after IPs were analyzed by WB using antibodies as indicated. B. eIF4E co-immunoprecipitated with SFs in the nuclear fractions of NOMO-1 cells. IPs were carried out using eIF4E overexpressing NOMO-1 nuclear lysates and anti-eIF4E antibody (eIF4E-IP) or appropriate IgG control (IgG-IP). IP samples were analyzed by WB as above and carried out in three biological replicates.C. eIF4E interaction with splicing factors depends on the presence of RNAs. The eIF4E IP was divided into two and treated with buffer or RNase. After elution, 2% of the nuclear extract and the IPs were resolved on SDS-PAGE and analyzed by western blotting with indicated antibodies. Representative gels of three biological replicates are shown (middle panel) and intensities measured using FIJI are shown in scatter plot (bottom panel) generated in PRISM with means depicted as horizontal lines and standard deviation as vertical lines. Each point represents an independent experiment. Fraction purity was assessed with the indicated antibodies (C top panel). Data information: (C, The number of independent experiments for each target is shown which varies from n=3-4 replicates. Error bars representative standard deviations (vertical line).
Figure 6C. Survival analyses for of High-eIF4E patients and Normal-eIF4E patients for which specimens were studied The rest of the cohort is shown for comparison. Log Rank test was performed and shows a significant difference in survival between the High-eIF4E group (10 specimens) and the rest of the cohort (432 specimens), as well as between High-eIF4E and Normal-eIF4E groups (11 specimens) (p-value < 0.05)
Figure 7C. Validation of splicing targets identified by rMATs in U2OS (upper panel) and NOMO-1 (lower panel) cell lines. RT-qPCR analysis using specific primers for each splicing event normalized to the corresponding total levels of that given transcript Data were normalized to Vector control to calculate fold change. The mean and standard deviation, as well as p-values were derived from three biological replicates. P-values were calculated between eIF4E overexpressing and Vector cells (when the asterisk is over eIF4E), and eIF4E cells treated with Ribavirin vs eIF4E overexpressing untreated cells (for the asterisk over Ribavirin). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001D. eIF4E associates with both pre-mRNA splicing substrates and their spliced products. The enrichment of mRNAs for splicing targets in eIF4E-RIPs versus input mRNAs from the nuclear fractions of 2FLAG-eIF4E U2OS cells monitored by RT-qPCR using primers for the specific splice event (S for "spliced"), pre-mRNA specific primers to the relevant intron or intron-exon boundary (NS for "unspliced"), and primers specific to common regions present in all spliced and not-spliced mRNAs (All). Data were normalized to input samples and presented as a fold change. The mean, standard deviation and p-values (Student t-test) were derived from three independent experiments (each carried out in triplicate). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001
Figure 1(F-K') (F', G', H', I') are the magnified images of the square region outlined in (F, G, H, I). (F-K) are merged images, (F'-K') are single channel images of (F-K) depicting the presence of NBs (Mira+). Clones (green) are outlined by dotted lines. All images in this figure, and all following figures are single confocal sections. (F-F' and G-G') No Mira+ NBs are recovered in deep sections of control or UAS-dpn clones at 16 hr after clone induction. Quantified in E. (H-H' and I-I') At 24 hr after clone induction, Mira+ NBs are recovered in UAS-dpn but not control clones. Quantified in E. (J-J'and K-K') At 72h after clone induction, numerous Mira+ NBs are recovered in UAS-dpn but not in control clones. Quantified in E.
Figure 2(C-L') Representative images of the deep medulla neuronal layer or NB superficial layer in the larval optic lobe, in which UAS-dpn or control are driven in clones by hs flp (marked by GFP and outlined) and stained with the stem cell marker, Miranda (Mira, magenta), and various temporal transcription factors (tTFs) (grey). (C-G') In superficial sections of control clones, Mira+ superficial NBs express the tTF: Hth, Ey, Slp, D, Tll in concentric patterns. Quantified in M. (H- L') In deep sections of the NB clone induced via UAS-dpn, Mira+ NBs do not express early tTF Hth or late tTF Tll, but are positive for mid tTFs Ey, Slp and D. Quantified in M.(M) Quantification of volume of cells that express a specific tTF as % of total Mira+ NB volume within a clone. Hth (Control n= 5, m= 31.86 ±3.89, UAS-dpn n= 6, m= 7.779 ±1.212), Ey (Control n= 7, m= 26.05 ±5.302, UAS-dpn n= 5, m= 64.08 ±4.936), Slp (Control n= 7, m= 34.87 ±2.753, UAS-dpn n= 9, m= 64.72 ±6.045), D (Control n= 4, m= 34.57 ±15.49, UAS-dpn n= 10, m= 45.2 ±6.339), Tll (Control, n= 5 m= 15.65 ±4.036, UAS-dpn n= 6, m= 3.584, ±1.87).
Figure 3(B) Quantification of % of Slp+ cells in Mira+ cells in control and UAS-dpn clones (expressed as the ratio between Slp+ volume and total Mira+ volume within a clone). 24 hr (Control n= 7, m= 14.25 ±2.696, UAS-dpn n= 9, m= 60.66 ±5.384), 48 hr (Control n= 3, m= 6.096 ±1.509, UAS-dpn n= 8, m= 43.64 ±3.398), 72 hr (Control n= 6, m= 35.13 ±3.242, UAS-dpn n= 9, m= 64.72 ±6.045), 96 hr (Control n= 6, m= 29.85 ±4.951, UAS-dpn n= 10, m= 58.2 ± 6.603).(C-F'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which UAS-dpn or control are driven by hs flp (marked by GFP and outlined) and dissected and stained with the stem cell marker, Miranda (Mira, magenta), and Sloppy-paired (Slp, grey). At 24h and 48h after clone induction, Mira+ NBs express mid tTF Slp compared to superficial NB control, quantified in B. (C'', D'', E'', F'') are the magnified images of the square region outlined in (C', D', E', F'), respectively.(G-J') At 96h after clone induction, Mira+ NBs express mid tTF Slp and do not express late tTF Tll, compared to control, quantified in B and K. (K) Quantification of % of Tll+ cells in Mira+ cells in control and UAS-dpn clones (expressed as the ratio between Tll+ volume and total Mira+ volume within a clone). Control n= 4, m= 48.34 ±5.956, UAS-dpn n= 5, m= 4.805 ±2.375. D
Figure 4Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe, in which UAS-dpn or control (UAS-luciferase) are driven by eyR16F10-GAL4 and stained with the stem cell marker, Miranda (Mira, magenta), (B-E) Mira+ NBs are recovered in superficial sections of control under eyR16F10-GAL4 expression at 6 hr APF and 10 hr APF but not from16 hr APF onwards, quantified in J. (F-I) Mira+ NBs are recovered in deep sections of UAS-dpn driven by eyR16F10-GAL4 at 6 hr APF, 10 hr APF, and 16 hr APF but not at 24 hr APF, quantified in J. Arrow in H points to the NBs. (J) Quantification of % Mira+ cells within eyR16F10-GAL4 expression domain in control and UAS-dpn (calculated as the ratio of Mira+ cell volume as a percentage of total eyR16F10-GAL4 domain volume). 6 hr APF (Control, n= 3, m= 14.82 ±3.521, UAS-dpn, n= 6, m= 18.13 ±1.46), 10 hr APF (Control n= 4, m= 11.65 ±1.605, UAS-dpn n= 5, m= 19.11 ±4.609), 16 hr APF (Control n= 4, m= 0.8897 ±0.239, UAS-dpn n= 7, m= 16.64 ±3.065), 24 hr APF (Control n= 4 m= 1.174, ±0.7226, UAS-dpn n= 6, m= 0.8518 ±0.4849).Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe, in which UAS-dpn or control (UAS-luciferase) are driven by ok107-GAL4 (outlined) and stained with the stem cell marker, Miranda (Mira, magenta), and mid-temporal progeny marker, Toy-GFP (grey). (K-L') More Toy+ progeny (grey) are present within the ok107-GAL4 domain (green, outlined) in UAS-dpn compared to control.(O-R') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which heat shock induced UAS-dpn or control (UAS-luciferase) clones are stained with the stem cell marker, Mira (magenta), late neuronal marker Tll (grey) or glial marker Repo (grey). Arrow points towards the small band of Tll+ neurons in control clones. (O-P') At 72 hr after clone induction, there is less Tll expression within UAS-dpn clones than control clone, quantified in S. (Q-R') At 72 hr after clone induction, there is less Repo expression within UAS-dpn clones than control clones, quantified in T.
Figure 6Representative images of deep medulla neuronal layer in the larval optic lobe, in which UAS-luc; UAS-dpn and UAS-D; UAS-dpn are expressed in clones by hs flp, and stained with the stem cell marker, Miranda (Mira, magenta), and/or Slp, Tll or Repo (grey). (A-B'') At 72 hr after clone induction, Mira+ NBs within UAS-luc; UAS-dpn clones express Slp; Mira+ NBs within UAS-D; UAS-dpn clones do not express Slp, quantified in I. (C-D') Representative images of deep medulla neuronal layer in the larval optic lobe, in which UAS-dpn and UAS-D; UAS-dpn are expressed in the ok107-GAL4 expression domain, and stained with the stem cell marker, Mira (magenta), and mid-temporal progeny marker, Toy-GFP (grey). Fewer Toy+ cells were recovered within the ok107-GAL4 domain (outlined) in UAS-D; UAS-dpn compared to UAS-dpn, quantified in E. (E) Quantification of % Toy+ cells within ok107-GAL4 expression domain in UAS-dpn and UAS-D; UAS-dpn (calculated as the ratio of Toy+ cell volume as a percentage of total ok107-GAL4 domain volume). UAS-dpn n= 9, m= 22.78 ±4.174, UAS-D; UAS-dpn n= 5, m= 14.42 ±2.394.(J-K) Quantification of % Tll+ or Repo+ cells within UAS-dpn clones compared to UAS-D; UAS-dpn clones (calculated as the ratio of Tll+ or Repo+ cell volume as a percentage of total clone volume). Tll (UAS-dpn n= 7, m= 0.7198 ±0.2389, UAS-D; UAS-dpn n= 9, m= 35.22 ±4.917), Repo (UAS-dpn n= 7 m= 1.488 ±0.2368, UAS-D; UAS-dpn n= 9, m= 14.13 ±2.672).(L-M) Mira+ NBs are present in deep sections of UAS-dpn driven by eyR16F10-GAL4 at 126h but are absent in UAS-D; UAS-dpn, quantified in N. (N) Quantification of % Mira cells within eyR16F10-GAL4 expression domain in UAS-dpn and UAS-D; UAS-dpn (calculated as the ratio of Mira+ cell volume as a percentage of total eyR16F10-GAL4 domain volume). UAS-dpn n= 6, m= 18.13 ±1.46, UAS-D; UAS-dpn n= 8, m= 1.859 ±0.4215. D
Figure 7(A-C'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc) and UAS-dpn and UAS-D; UAS-dpn are expressed in hs flp clones (72 hr after clone induction). Miranda (Mira, magenta) and EdU (grey). Despite increased number of Mira+ cells in UAS-dpn compared to control, each NB underwent slower cell cycle progression, as indicated by EdU+ incorporation. Quantified in E. This slow cell cycle speed was not rescued in UAS-D; UAS-dpn clones. Quantified with UAS-luc; UAS-dpn in F.(G-H''') Representative images of the deep medulla neuronal layer in the larval optic lobe, where UAS- E2f1; UAS- CycE; UAS-dpn or UAS-dpn are expressed in the ok107-GAL4 domain (outlined), and stained with the stem cell marker, Mira (magenta), mid-tTF, Slp (grey) and late progeny marker, Repo (Cyan). Quantified in I-K. UAS-dpn NBs express Slp and do not create Repo+ progeny, whereas UAS- E2f1; UAS- CycE; UAS-dpn NBs do not express Slp and create Repo+ progeny.
Figure 8(B-D''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc), UAS-NACT, and UAS-D; UAS-NACT are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Sloppy paired (Slp, grey), Tailless (Tll, cyan). UAS-NACT NBs express Slp and not Tll. UAS-D; UAS-NACT NBs express Slp and Tll, quantified in E-H.(J-K') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which UAS-NACT and UAS-NACT; UAS-dpn are driven by ok107-GAL4 (marked by GFP and outlined) and stained with the stem cell marker, Mira (magenta), and mid-temporal progeny marker, Toy-GFP (grey). Fewer cells express Toy+ in UAS-NACT; UAS-dpn compared to UAS-NACT, quantified in I.
Figure 9(A-C''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-lacz), lola-Ri, and UAS-D; lola-Ri are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Sloppy-paired (Slp, grey), Twin of eyeless (Toy, cyan). lola-Ri induces ectopic Mira+ NBs in the deep medulla layers, compared to control, quantified in E.(H-J''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-lacz), lola-Ri, and UAS-D; lola-Ri are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Tailless (Tll, grey), Reversed polarity (Repo, cyan). lola-Ri NBs express less Tll compared to control. There are fewer Tll+ and Repo+ progeny in the eyR16F10-GAL4 domain in lola-Ri compared to control. UAS-D; lola-Ri NBs express more Tll as well as more Tll+ and Repo+ progeny in eyR16F10-GAL4 domain compared to lola-Ri alone, quantified in K-L.
Figure 1A IMR90 ER:RasV12 primary fibroblasts were cultured in the presence of EtOH (proliferating) or 100nM 4OHT (for 6-8days to induce RasV12 expression and oncogene-induced senescence, OIS). mRNA levels of 4OHT cells, normalised to PUM1 and presented relative to EtOH control. (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)B, C Cells as in A were analysed by Western blot for Lamp1, p62 and Cathepsin B and quantified, normalised to GAPDH and expressed relative to EtOH. (n=3 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001D, E Cells as in A were fixed and immunostained for Lamp1 and signal intensity was quantified. Scale bars: 10µm (n=3 independent experimental repeats (at least 40 cells analysed per condition, from at least 4 fields of view, per repeat). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)K, Cells as in A were incubated with DQ-BSA and TR-BSA for 2 hours, fixed and imaged by confocal microscopy. Scale bar: 20µm (n=3 independent experimental repeats (at least 240 cells analysed from 8 fields of view per condition).O, Cells as in A were incubated with 50µg/ml cycloheximide (CHX) for the timepoints indicated. Degradation of autophagy receptors, p62 and NDP52 was assessed by Western blot (n=3 independent experimental repeats).
Figure 2A Representative images of GFP-TFEB localisation in EtOH and 4OHT-treated fibroblasts, in full nutrient conditions. Scale bar: 20µm.B, EtOH and 4OHT-treated fibroblasts immunostained for endogenous TFE3 in the conditions indicated; starvation indicates serum starvation overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. Scale bar: 20µm; (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat).C EtOH and 4OHT-treated fibroblasts in the conditions indicated; starvation indicates serum starvation overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. % cells with nuclear TFE3 was quantified. (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat). Data information: Violin blots include all datapoints from n=3, lines represent median and upper and lower quartiles; Analysis: one-way ANOVA with Tukey's multiple comparison test * p<0.05; ** p<0.01; *** p<0.001.
Figure 3D Cells treated as in C were analysed by Western blotting.H Representative images of cells treated as in C,D before being fixed and immunostained with antibodies against Lamp2 and p62. Scale bar: 20µm
Figure 4A Fibroblasts stably expressing GFP-TFEB were treated 4OHT to induce senescence (or EtOH control). Cells were lysed and subject to Western blotting for antibodies as indicated. B Quantification of A. (n=4 independent experimental repeats) C Quantification of A. (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. B and C: analysed by 2-tailed, unpaired T-Test * p<0.05; ** p<0.01; *** p<0.001.D Western blot of GFP immunoprecipitation of control (EtOH) and senescent (4OHT) cells expressing GFP or GFP-CDK4 and FLAG-TFEB.H Fibroblasts stably expressing GFP-TFEB were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. I Quantification of H (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)J Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. K, L Quantification of J (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)M Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were fixed and immunostained with antibodies against p62 and Lamp2, Scale bar: 20µm.O Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were incubated with serum-free media overnight which was collected and subject to ELISA analysis for IL-6 (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)