EMBO/soda-nel huggingface dataset:
TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes (2022)
Figure 6

Figure 6. SERBP1 preserves ribosomes in human cells.

A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.

D-E. Polysome profile analyses of HEK293T cells treated with control siRNA (siCtrl) (D) or SERBP1 siRNA (siSERBP1) (E), with or without INK128 treatment for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.

F. Ribosome subunit analysis of HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) under normal conditions. Minimum of four biological replicates are analyzed.

G-H. Ribosome subunit analysis (G) and its quantification (H) of HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.

I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.

K. In vitro kinase assay using recombinant mTORC1 and purified SERBP1 as a substrate in the presence of 32P-γ-ATP. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used to inhibit mTOR.

L. Model showing the TORC1-mediated regulation of dormant 80S ribosome formation and translation recovery upon refeeding via Stm1/SERBP1.

Extended View Figure legends
Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity (2020)
Figure 1

Fig 1. TRIM21 uses anti-N antibodies to protect against LCMV infection. A-F) Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Weights (A) and Kaplan-Meier survival (B) were compared over 15 days post infection (pi). C) Sera from WT mice was analysed for N protein (N) and glycoprotein (GP)-specific antibodies by ELISA, each data point corresponds to one mouse from the same experiment. D) N-specific antibody ELISA was used to compare antibody responses in sera of WT and KO mice. E-F) In vitro neutralisation experiments were performed with sera collected day 8pi from WT, KO and UI mice. E) Sera was pre-incubated with LCMV, then the virus-serum mix was added to MEF cells and LCMV infection titre after 16 hours was measured by FFA. F) Sera was electroporated into MEFs, then cells were plated in triplicate and LCMV was added 4 hours later. LCMV infection titre after 16 hours was measured by FFA (three replicates). G) Anti-N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA. H) Anti-N mAb KL53 was co-electroporated with recombinant N protein into WT and KO MEFs, and immunoblotting for N was performed after 3 hours. Electroporation of recombinant KL53 expressing the TRIM21 non-binding mutation H433A was unable to mediate N protein degradation.

Data information: All data is presented as mean with standard error, * p <0.05, unpaired t-test.
Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity (2020)
Figure 2

Fig 2. Non-neutralizing anti-N antibodies reduce viraemia but require TRIM21. A-B) Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). A) Weights were monitored throughout infection, with final day 8 weights of individual mice presented separately. B) Viral titres in the spleen, liver, lung and kidney of all mice were determined by FFA day 8pi. Each data point represents one mouse, with results from two repeat experiments combined.

Data information: All data is presented as mean with standard error, ** p<0.01, *** p<0.001, NS not significant, unpaired t-test.
Viral nucleoprotein antibodies activate TRIM21 and induce T cell immunity (2020)
Figure 4

Fig 4: N-specific CTLs induced by anti-N antibodies and TRIM21 drive potent in vivo cell killing. A) Splenocytes from uninfected CD45.1 mice, either pulsed with 3 concentrations of N peptide and cell trace violet (CTV) or unlabelled control cells, were transfused intravenously into WT and KO mice (CD45.2) that had been infected with 0.5 x 10^5 FFU LCMV 8 days earlier. After 3 hours, spleens from recipient mice were harvested and the proportion of CTV-labelled CD45.1 cells was analysed by flow cytometry. Histograms from single representative uninfected (UI), WT and KO mice are presented, showing the proportion of CD45.1 cells remaining for each of the labelled fractions normalised to mode. Summary data from all individual mice in the same experiment is presented in associated scatter plot, showing the mean ± standard error. B) Labelled splenocytes as for (A) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier, and received mAb KL53 on days 1 and 3pi. Flow cytometry histograms from single representative mice of each genotype. Summary data from all mice in the experiment is presented, showing the mean ± standard error.

Data information: Horizontal bars on each graph correspond to the mean ± standard error, ** p<0.01, *** p<0.001, unpaired t-test.
Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage (2020)
Figure 1

Figure 1. Inhibition of guanosine nucleotide synthesis elicits distinct p53 responses.

A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.

B, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate.

C-D, Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D).

E, Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA, in presence of dimethyl sulfoxide (DMSO) or 400 µM guanosine. GAPDH was used as a loading control.

Data information: In panels B, C and D, data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.
Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage (2020)
Figure 2

Figure 2. The IRBC and ATR distinctly regulate p53 stabilization upon nucleotide depletion.

A, HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone (-) or the indicated concentration of MPA for 24 hrs. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel).

B, HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel).

C, HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence (-) or the presence (+) of the combination of 10 µM of the ATR inhibitor VE-821 (ATRi) and 10 µM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (-) or MPA 10 µM for additional 24 hrs. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel).

Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.
Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage (2020)
Figure 3

Figure 3. Protein stability of p21, but not formation of the IRBC complex, is reduced by higher concentrations of MPA.

A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA.

B, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section.

C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates from (B).

D-E, HCT116 cells were treated as in (B) for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG).

D, Levels of HDM2, RPL5, RPL11, and the IP negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5 and RPL11 of at least two independent experiments is shown (lower right panel).

E, the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates.

F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment.

G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel.

Data information: In panels A, E and G, data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.
Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage (2020)
Figure 4

Figure 4. S-phase entry and inhibition of DNA replication are induced at high concentrations of MPA.

A, HCT116 were treated as indicated for 24 hrs, subjected to propidium iodide staining and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments.

B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells.

C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means.

D, Representative images from (C).

E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm.

F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.
Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage (2020)
Figure 5

Figure 5. The downregulation of p21 facilitates S-phase entry at high concentrations of MPA.

A, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels.

B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.
Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage (2020)
Figure 6

Figure 6. Disruption of the IRBC enhances MPA-mediated DNA-damage.

A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments.

B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control.

C and D, Statistical analysis of the immunostaining of γ-H2AX (C) or 53BP1 (D) in HCT116 cells treated as in (A) (see Methods). Total nuclear intensities were obtained on >6000 (γ-H2AX) and >1000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.

E, Representative images from (C-D) of cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μm

F, HCT116 cells treated as in (B) were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test
Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3 (2022)
Figure 1

Figure 1. OTUD1 is up-regulated in aortic endothelium of Ang II-induced mice. (A) mRNA levels of members of OTU family in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n = 5 biological replicates). (B-C) Western blot analysis (B) and densitometric quantification (C) of OTUD1 in aortas of control and Ang II-induced mice. β-actin was used as the loading control (n=5 biological replicates). (D) mRNA levels of OTUD1 in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n=5 biological replicates). (E) Representative immunofluorescence staining of CD31 (green) and OTUD1 (red) of aortic sections (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and OTUD1 (red) of aortic sections (scale bar=25 μm). (G-H) Time-course of OTUD1 induction in response to Ang II in HUVECs. HUVECs were exposed to 1 μM Ang II for indicated time. Western blot analysis (G) and densitometric quantification (H) of OTUD1 were shown (n = 3 biological replicates). GAPDH was used as the loading control. Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3 (2022)
Figure 2

Figure 2. OTUD1 deletion attenuates vascular remodeling and collagen deposition through inhibiting EndMT in Ang II-induced mice. (A) Representative images of H&E staining of aortic sections of the mice from four groups (scale bar=25 μm). E, endothelium; M, media; A, adventitia. (B) The quantification of arterial wall thickness in panel A; n=5 biological replicates. (C) Representative images of Masson's Trichrome staining of aortic sections of the mice (scale bar=50 μm). (D) The quantification of collagen deposition in panel C; n=5 biological replicates. (E-F) Western blot analysis (E) of VE-cadherin, Vimentin, Twist, Snail, and OTUD1 and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues of the mice. β-actin was used as the loading control (n=5 biological replicates). (G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues of the mice were examined by real-time qPCR assay. The values were normalized to Rn18s (n=5 biological replicates). Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3 (2022)
Figure 3

Figure 3. OTUD1 exacerbates Ang II-induced EndMT in HUVECs. (A) HUVECs were transfected with OTUD1 siRNA (si-OTUD1) for 24h, while the control cells were transfected with negative control (NC) siRNA. Levels of OTUD1 protein were measured by Western blot (n=3 biological replicates). (B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control. (C) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. mRNA levels of Cdh5, Vim, Acta2, Twist1, Snai1, Tgfb1 and Col1a1 were determined by RT-qPCR (n=3 biological replicates). The values were normalized to β-actin. (D) HUVECs were transfected with Flag-OTUD1 plasmid for 24 h. Control cells were transfected with empty vector (EV). Levels of OTUD1 were measured by Western blot (n=3 biological replicates). (E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control. (F) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. mRNA levels of Cdh5, Vim, Acta2, Twist1, Snai1, Tgfb1 and Col1a1 were determined via RT-qPCR (n=3 biological replicates). The values were normalized to β-actin. Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.
Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3 (2022)
Figure 4

Figure 4. OTUD1 associates with the MH2 domain of SMAD3 and reverses the K48- and K63-linked ubiquitination of SMAD3 via cysteine 320 on OTUD1. (A) Schematic illustration of quantitative proteomic screen to identify proteins binding to OTUD1. (B) MS/MS spectrum of the peptide showing TVDGFTDPSNSER from SMAD3. (C) Co-immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag-OTUD1. Exogenous Flag-OTUD1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G. (D) Co-immunoprecipitation of OTUD1 and SMAD3 in 293T cells co-transfected with Flag-OTUD1 and HA-SMAD3. OTUD1 and SMAD3 were immunoprecipitated by anti-Flag and anti-HA antibodies, respectively. IgG, immunoglobulin G. (E) The protein structure of SMAD3 and the schematic illustration of SMAD3 domain deletion constructs. (F) Identification of the OTUD1-binding domain of SMAD3. Plasmids encoding GFP-labeled SMAD3, or three GFP-labeled SMAD3 mutants (mut-SMAD3), respectively, and Flag-OTUD1 were transfected in 293T cells for 24h. The interactions between OTUD1 and SMAD3 or SMAD3 mutants were determined by co-immunoprecipitation using anti-Flag and anti-GFP antibodies. (G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.