EMBO/soda-nel huggingface dataset:
A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB (2012)
Figure 4

mTORC1 binds and phosphorylates TFEB on the lysosomal surface. (A) Spinning disk confocal image of a MEF cell that co‐expresses TFEB–GFP and mRFP–Rab7 (green and red in the merge, respectively). (B) Time‐lapse of TFEB‐ and Rab7‐positive lysosomes from the boxed region in (A). Time intervals are in seconds. (C) Time‐lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB–GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1‐induced lysosomal accumulation of TFEB–GFP. Time intervals are in minutes. (D) Immunofluorescence of HEK‐293T cells expressing TFEB–3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (E) FRAP analysis of TFEB–GFP‐positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots. (F) Time‐lapse of photobleaching and fluorescence recovery of TFEB–GFP‐positive lysosomes from control‐treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds. (G) Torin 1 increases binding of TFEB to mTORC1. HEK‐293T cells that express TFEB–3 × FLAG along with HAGST‐Rap2A or HAGST‐RagsDN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG–Metap2 served as negative control. (H) Immunofluorescence of HEK‐293T cells that express TFEB–3 × FLAG along with Rap2A (top) or the RagsDN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.
A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB (2012)
Figure 5

Rag GTPases control TFEB subcellular localization. (A–C) Immunofluorescence of HEK‐293T cells that express TFEB–3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (D) Quantification of the number of cells with nuclear TFEB from each condition in (A–C). (E, F) Immunofluorescence of HEK‐293T cells that express TFEB–3 × FLAG along with Rap2A (E) or the RagsCA mutants (F), subjected to the indicated treatments and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (G) Quantification of the number of cells with nuclear TFEB from DMSO‐ and CQ‐treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.
A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB (2012)
Figure 6

The lysosome regulates gene expression via TFEB. (A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2‐month‐old Tcfebflox/flox (control) and Tcfebflox/flox;Alb‐Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies. (B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t‐test (two tailed) *P‐value ⩽0.05.
Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation (2020)
Figure 1

Figure 1. eEF3 is essential for tri- & polypeptide formation

(A) Schematic of the translation elongation cycle as studied here.

(B) Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A-GTP-[14C]Phe-tRNAPhe in the absence (orange, 0.13±0.02 s-1) or presence (cyan, 0.15±0.02 s-1) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates.

(C) Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15±0.01 s-1), eEF2 (red) or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates.

(D) Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4±0.14 s-1), eEF2 (red, 1.0±0.5 s-1) or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3.

Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1.
Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation (2020)
Figure 2

Figure 2. eEF3 in tRNA movement during translocation and E-site clearance

(A) Schematic of tRNA translocation.

(B) mRNA-tRNA translocation monitored in a time-resolved Pmn assay upon mixing 80S 2C with Pmn in the absence (brown triangles) or presence of eEF2 and eEF3 (green, 0.13±0.01 min-1), eEF2 (red, 0.030±0.005 min-1) or eEF3 (blue, 0.10±0.01 min-1). Data are normalized to Met-Phe-Pmn formation in presence of eEF2 with the maximum value set to 1. Data presented as mean ± SEM of n = 3.

(C) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.

(D) tRNAfMet(Flu) co-eluting with 80S 2C incubated without factors (orange), with eEF2 (red), with eEF2 and eEF3 (magenta), or with eEF2, eEF3 and TC-Val (green) for 15 min before loading on a BioSuite 450 size-exclusion column to separate ribosome-bound and -unbound tRNA. Data are normalized to tRNAfMet(Flu) bound to 80S 2C in the absence of eEF2/eEF3 with the maximum value set to 100%. Data presented as mean ± SEM of n = 3.

(E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.
TSG101 Associates with PARP1 and is Essential for PARylation and DNA Damage-induced NF-κB Activation (2022)
Figure 2

Figure 2. TSG101 is Essential for the DNA damage-induced NF-κB Pathway

A: IR-induced NF-ĸB pathway activation was analyzed via luciferase activity of HEK-Luc cells, transfected with the indicated siRNAs. The indicated cells were irradiated (20 Gy, 8 hours before analysis). A late time point was used for efficient luciferase expression. Luciferase activity was normalized to the viable cell number (TOX fluorescence). The data represent three biological and five technical replicates. Error bars represent mean ± SD. For knockdown efficiency, see Fig EV2A. The conditions were compared with an ordinary one-way ANOVA (****p < 0.0001).

B: U2-OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 µM, 90 min. before analysis). Whole-cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.

C: U2-OS cells were transfected with the indicated siRNAs and exposed to irradiation (20 Gy, 90 min. before analysis). The expression of indicated genes was analyzed using RT-qPCR. The mRNA expression was normalized to the expression of the housekeeping genes ACTA1, RPL13A, and TBP2. Data represent three biologically independent experiments +/- SEM. The conditions were compared with an ordinary one-way ANOVA (*p < 0.05; **p < 0.01, ***p < 0.001).

D: U2-OS cells were transfected with non-targeting (mock) or TSG101-targeting siRNAs and irradiated (20 Gy) 90 min. before analysis. Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one-way ANOVA (*p < 0.05) in the right panel. Error bars represent mean ± SD
TSG101 Associates with PARP1 and is Essential for PARylation and DNA Damage-induced NF-κB Activation (2022)
Figure 3

Figure 3. TSG101 Controls PARylation

A: Nuclear-cytoplasmic fractionation of U2-OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 µM, 24 hours before analysis). Cytoplasmic export of p-ATM was induced by etoposide (50 µM, 45 minutes before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH-A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1) (Jungmichel et al, 2013).

B: U2-OS cells were transfected with non-targeting (-) or TSG101-targeting (+) siRNAs and cells were irradiated (+) or not (-) as indicated (20 Gy) 10 min. before analysis. Whole-cell extracts were immunoblotted with the 10H PAR monoclonal antibody.

C: siRNA-transfected U2-OS cells as in B were etoposide-treated or not, as indicated. Whole-cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly- and mono-(ADP-ribose). Note that PARylation also occurred during cell lysis in untreated cells.

D: U2-OS cells were transfected with non-targeting or VPS28-targeting siRNAs. Whole-cell extracts were immunoblotted with indicated antibodies.

E: Total mRNA was extracted from the samples in D. Expression of TSG101 was analyzed by RT-qPCR and normalized to two housekeeping genes, ACTA1 and RPL13A. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05). Error bars represent mean ± SEM.

F: Cells were irradiated (20 Gy) as indicated 10 min before analysis. Whole-cell extracts were immunoblotted with the indicated antibodies.

Data information: The results are each presentative for three (panels A-D and F) or six (panel E) independent experiments.
TSG101 Associates with PARP1 and is Essential for PARylation and DNA Damage-induced NF-κB Activation (2022)
Figure 4

Figure 4. TSG101 Interacts with PARP1

A: Proximity ligation assay (PLA) assay with unstimulated, etoposide (Eto)-treated (50 µM, 10 min. before analysis), or etoposide plus olaparib-(Olap) co-treated (50 µM of etoposide, 10 min. before analysis and 10 µM of olaparib 24 hours before analysis) U2-OS cells. The red dots represent PARP1-TSG101 interaction. The nucleus is stained with DAPI (blue). Scale bar: 10 µm. As negative controls, PLA was performed without primary PARP1 or TSG101 antibodies. As an additional control, PLA was performed in PARP1 depleted cells to show specificity of the PLA signal. The data is representative of three biologically independent experiments.

B: Quantification of PLA dots per nuclei in A. Blind counting was performed from 30 replicates three biologically independent experiments. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001). Error bars represent mean ± SD.

C: U2-OS cells were cotransfected with the indicated deletion constructs of mCherry-tagged TSG101 and EGFP-tagged PARP1. Immunoprecipitation of GFP was performed using whole-cell extracts.

D: Diagram showing summary of PARP1 interacting and non-interacting TSG101 domain deletion mutants.

E: U2-OS cells were transfected with the indicated, coiled-coil containing CC+SB and coiled-coil deleted ΔCC constructs. Cells were irradiated (20 Gy, 5 minutes before analysis) and the decomposed NADH and total NAD+ levels were quantified. The NADH/NAD+ ratio is shown in the right panel. The conditions were compared with unpaired t-test (*p<0.05). Error bars represent mean ± SD.

F: In-vitro PARylation reactions were carried out with the indicated recombinant and purified proteins, including 8 ng PARP1 in the presence of sssDNA (sheared salmon sperm DNA) and MgCl2. 100 ng of TSG101 was added to the reaction, as indicated. The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls.
TSG101 Associates with PARP1 and is Essential for PARylation and DNA Damage-induced NF-κB Activation (2022)
Figure 5

Figure 5. PARP1 is Trapped in DNA Lesions in the Absence of TSG101

A: Schematic of the live-cell PARP1 recruitment assay. U2-OS cells transfected with GFP-tagged PARP1 were sensitized with DAPI (10 µg/ml, for 10 min.). After 405 nm microirradiation, PARP1 recruitment to and dissociation from the DNA lesion was recorded over time.

B: Representative images of PARP1-GFP association with laser-microirradiation sites in untreated (mock), olaparib-treated (10 µM, 24 hours before analysis) or siTSG101-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 7.5 µm. The image is representative of nine replicates from three biologically independent experiments. Go to Movies EV1-3 for further illustration.

C: Kinetics of PARP1-GFP recruitment and dissociation from DNA lesions were measured at times indicated. Nine nuclei were analyzed for each indicated condition from three biologically independent experiments. The data are shown as mean GFP intensity in the microirradiated area ± SEM, normalized to the mean GFP intensity of corresponding whole nuclei. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; ***p < 0.001).

D: Relative PARP1-GFP recruitment to DNA lesions for each indicated condition at 12 min. post-irradiation. The conditions were compared with an ordinary one-way ANOVA (**p < 0.01). Error bars represent mean ± SEM.

E: Scheme of indirect immunofluorescence analysis of DNA damage sites. U2-OS cells were GFP-PARP1-transfected and microirradiated as in A. Cells were fixed shortly after rapid PARP1 recruitment to the DNA lesions (1 min post-microirradiation) and stained for TSG101 by indirect immunofluorescence.

F: Indirect immunofluorescence of TSG101 at indicated times post damage. DNA lesions were generated as in B. Stippled lines indicate the applied laser beam. Recruitment of PARP1 to DNA lesions was recorded from live cells. Cells were fixed at 1 min post-microirradiation and stained for TSG101. For specificity of the TSG101 staining see Fig EV5A. Scale bar: 10 µm.

G: Representative images of PARP1-GFP association/dissociation at laser-microirradiation sites in untreated (mock) or siUBAP1-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 10 µm. The image is representative for four independent experiments.
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 1

Figure. 1. Nintedanib/BIBF1120 prevents MAPKi-induced ECM remodeling, decreases resistance to targeted therapy and delays tumor relapse. (A-G) Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (B) Representative median graphics showing tumor growth following treatment (n = 6). Two-way ANOVA was used for statistical analysis. ** P≤0.01. (C) Kaplan-Meier survival curves of mice treated with the indicated therapies (n = 6). Log rank (Mantel-Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P≤0.0001. (D) Mouse body weight was measured at the indicated times. Data shown are mean±SD (n = 6). (E-F) Tumor sections were stained with picrosirius red and imaged under polarized light. (E) Representative image of collagen fibers network in tumors from mice under the different treatments. Scale bar 200 μm. (F) Quantification of collagen fibers thickness (n = 6 for control, BIBF and BRAFi/MEKi groups and n = 5 for BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification. **P≤0.01, ***P≤0.001, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated. (G) Heatmap showing the differential expression of ECM and myofibroblast/CAF genes in mice treated with MAPK-targeted therapies with or without BIBF compared to control mice (log2 ratio, n = 5). (H) Heatmap and Western Blot showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells. Heatmap represents the mean of expression of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells treated with BIBF (2 µM, 72 h) or vehicle alone by RT-qPCR (n = 3). (J) Crystal violet viability assay of M238R cells treated with BRAFi/MEKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1 µM), BIBF (2 μM) or with BRAFi/MEKi (1 μM) plus BIBF (2 μM) for 72 h. Paired Student t-test was used for statistical analysis. ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated. Data is represented as mean ± SD from a triplicate representative of 3 independent experiments.
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 2

Figure. 2. Expression of miR-143/-145 is correlated with the undifferentiated mesenchymal-like MAPKi-resistant phenotype and is negatively regulated by Nintedanib/BIBF1120. (A) Heatmap showing the differential expression of a selection of miRNAs known as "FibromiRs" in human BRAFV600E mutant melanoma cells sensitive to MAPK-targeted therapies (M229, M238, M249) and the corresponding BRAFi-resistant cells. Expression of indicated FibromiRs was evaluated by RT-qPCR; log2 (Resistant vs Parental). Expression level of AXL and MITF phenotypic markers for each resistant cell line is indicated. (B) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control mice and mice treated with the indicated therapies (see legend of Fig.1 for details) was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001. (F, G) Phenotype switch/invasive/ECM markers (F) and relative miRNAs expression levels (G) were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.
Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma (2022)
Figure 3

Figure. 3. miR-143/-145 cluster promotes ECM reprogramming, melanoma cell dedifferentiation and drug resistance. (A) Heatmap showing the differential expression of a selection of ECM-related genes, cytoskeleton and myofibroblast markers in 3 distinct cell lines (M238P, UACC62P, M229P) transfected with the indicated mimics (control (miR-neg), miR-143 or miR-145 mimics, 72 h, 30 nM), assessed by RT-qPCR. (n = 3) (B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. (C) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in (A). (D) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of inhibitors as in (B). (E) Proliferation curves using time-lapse analysis of cells with the IncuCyte system. Graph shows quantification of cell confluence. Two-way ANOVA analysis was used for statistical analysis. ****P≤0.0001. (F) Migration assay performed in Boyden chambers. Representative images showing migrating cells in the different conditions. The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. * P≤0.05, ***P≤0.001. (G) Western Blot analysis of phenotype switch markers on lysates from M238P cells treated as in (A). (H) Crystal violet viability assay of M238P cells treated 72 h with the different combinations of inhibitors in presence or not of BRAFi (Vemurafenib, 3 μM). Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. ****P≤0.0001.