EMBO/soda-nel huggingface dataset:
A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death (2020)
Figure 6

Figure 6. MRP1 inhibitor MK-571 shifts cellular thiol pools, further potentiating APR-246 efficacy

Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n.

Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3).

Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h.

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.

Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).

Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).

Data information: Data are represented as mean ± SEM. See also Appendix Figure EV6 and Appendix Figure S6.
A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death (2020)
Figure 7

Figure 7. GSH and Cys availability determines APR-246/MQ accumulation and sensitivity to APR-246

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.

Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.

Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)

OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G.

Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).

Data information: Data are represented as mean ± SEM. See also Appendix Figure S7
In situ and high-resolution cryo-EM structure of a bacterial Type VI secretion system membrane complex (2019)
Figure 4

Figure 4. The TssJ' monomer and its function

A Ribbon diagram and locally sharpened surface representation (transparent= of the three TssJ protomers in orange, labelled TssJ.i, TssJ.o and TssJ' for the inner, outer and additional monomer respectively. In light green and blue are the TssM.i and TssM.o protomers respectively

B Superimposition of the ribbon diagrams of TssM-TssJ heterodimer within the inner (green-orange) and the outer (blue-orange) pillars

C Ribbon diagram of the TssM.o (outer pillar) with the TssJ.o and the TssJ' (orange)

D Fluorescence of prey cells (green) indicating their survival against attacker cells after 4 h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.

E Time-lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental (WT) and TssJ mutated strains (TssJ D97K). Individual images were taken every 60 seconds. The corresponding time of each micrograph is indicated in the lower right part of the image. Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads. Scale bars, 1 µm.
In situ and high-resolution cryo-EM structure of a bacterial Type VI secretion system membrane complex (2019)
Figure 5

Figure 5. The TssM protein and the periplasmic gate

A The high-resolution structure of MC complex in different orientations. TssJ is in orange, TssM is in green and blue according to their position as an inner or outer pillar respectively.

B Pseudoatomic model of the N-terminus of TssM.i (green), showing it is composed of 8 helices, coloured in different shades of red and numbered from the C-terminus (1) to the N-terminus (8).

C The pore radius formed by the inner pillar of TssM protomers is highlighted as dots and mapped using HOLE. The atomic model of TssM is coloured according to secondary structure (Helices in pink and strands in brown). The graph shows the pore radius calculated along the centre of the inner pillar of TssMs.

D Ribbon diagram of the region surrounding the periplasmic gate, with the amino acids involved in the formation of the gate in atom form. Gln779 and Asn780-781 from the inner pillars of TssM (green) are labelled in one of the protomers.

E Fluorescence of prey cells (green) indicating their survival against attacker cells after 4h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.

F Fluorescence microscopy recordings showing sfGFPTssM foci in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). TssM foci containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.

G Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). Fluorescent sheath containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.
Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction (2022)
Figure 3

Figure 3. Mutations in the active center render Nsp16 catalytically inactive.

(A) In vitro Methyltransferase activity. Recombinant proteins Nsp10 and Nsp16 wt or Nsp16mut were combined with cap0 RNA and Adenosyl-L-methionine ([methyl-3H] SAM) for 90 min at 37°C. Methylation of cap0 RNA was quantified using a scintillation counter (Beckmann).(-) control did not contain any recombinant protein. Results are plotted as mean of triplicate reactions with error bars representing SD. One out of three independent biological repeats is shown.

(B) MTase activity of Nsp16 upon viral infection. Cellular mRNA was purified 24 h postinfection with SARS-CoV-2 wt or Nsp16mut (triplicate infections, biological replicates). Purified mRNA was incubated with 100U of the recombinant MTase VACV VP39 (NEB) and [methyl-3H] SAM for 90 min at 37 °C. Methylation of mRNA from SARS-CoV-2 infected cells was quantified using a scintillation counter (DPM3H; Beckmann). (-) control did not contain cellular RNA. Results are plotted as mean of triplicate infections with error bars representing SD. One out of three independent experiments is shown (n=3).

Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.
Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction (2022)
Figure 4

Figure 4. Replication of Nsp16mut is highly sensitive to type I interferon.

(A) Calu-3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS-CoV-2 wt (WT) or Nsp16mut at 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates). One out of three independent experiments is shown (n=3). Three days postinfection, protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90.

(B) Calu-3 cells were incubated with medium (-IFN) or 100 U/well type I IFN at the indicated time points prior- or postinfection. Cells were infected with normalized amounts of SARS-CoV-2 wt (WT) or Nsp16mut at 0.015 RNA copies/cell. Viral load in the supernatant was quantified at 3 dpi by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates). One out of three independent experiments is shown (n=3).

Data information: Statistical analysis was done using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).
Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction (2022)
Figure 5

Figure 5. SARS-CoV-2 infection triggers an enhanced MDA5-dependent immune response in the absence of active Nsp16.

(A) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), or luciferase KO control cells (Ctrl) were infected with normalized amounts of SARS-CoV-2 wt (WT) with 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates).

(B) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), cGAS (cGAS KO), or control KO cells (Ctrl) were inoculated with normalized amounts of Nsp16mut with 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates).

(C) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), or control KO cells (Ctrl) were seeded into 96well plates and infected with increasing amounts of SARS-CoV-2 wt (WT) or Nsp16mut in triplicates (biological replicates). Type I IFN release was quantified after 48 h by incubating supernatants with HEK-Blue IFN-α/β reporter cells. SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD.

(D) Calu-3 cells (Ctrl) were seeded into 96 well plates and infected with 10 RNA copies/cell of SARS-CoV-2 wt (WT) or Nsp16mut in the presence of increasing amounts of Remdesivir (RDV). Type I IFN release was quantified after 48 h by IFN bioassay on HEK-Blue IFN-α/β and is depicted as mean of quadruplicates with error bars representing SD. In parallel, infectivity of SARS-CoV-2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT-qPCR targeting viral polymerase RdRp transcripts.

(E, F, G) Calu-3 cells were mock infected or infected with SARS-CoV-2 wt or Nsp16mut at 0.15 copies/cell. At 24 hpi, total RNA was extracted from lysed cells and expression of the indicated ISGs was quantified using RT-qPCR. ISG levels were normalized on GAPDH transcript expression. Results are plotted as mean of triplicate infections with error bars representing SD (biological replicates). One out of three independent experiments is shown (n=3).

Data information: Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).
Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms (2022)
Figure 2

Figure 2. Etoposide, but not doxorubicin, creates breaks and DNA-protein crosslinks during DNA replication

(A) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, etoposide, or doxorubicin. Purified DNA intermediates were digested with XmnI, which cuts the plasmid template once, to allow for identification of DNA breaks.

(B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography.

(C) Quantification of DNA breaks from (B). Mean ± SD, n=3 independent experiments. Abundance of breaks was calculated as a percentage of total lane signal (% of total).

(D) Quantification of linears ('Lins') from (B). Mean ± SD, n=3 independent experiments. Abundance of linears (AU) was normalized to the maximum recorded signal within each individual experiment.

(E) Plasmid DNA was replicated as in (A). Samples were split and either treated with ('+Prot K') or without ('-Prot K') proteinase K.

(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography.

(G) Quantification of DPC-containing molecules from (F). Mean ± SD, n=3 independent experiments. Abundance of the DPC-containing molecules was calculated as a percentage of total lane signal (% of total).

(H) Quantification of the amount of supercoiled circular monomers ('scCMs') from '+ProK' samples from (F). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers (AU) was normalized to the maximum recorded signal within each individual experiment.
Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms (2022)
Figure 3

Figure 3. Etoposide and doxorubicin inhibit DNA replication through different mechanisms

(A) Plasmid DNA was replicated in Mock- or TOP2α-immunodepleted extracts in the presence or absence of vehicle control, etoposide, or doxorubicin. In mock-depleted extracts (ΔMock), the final replication product are supercoiled circular monomers ('scCMs'). In TOP2α-depleted extracts (ΔTOP2α), the final replication product are catenanes ('Cats') because TOP2α is required for DNA unlinking.

(B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. Linear molecules, arising from DNA breaks, are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes. See also Fig EV3A-B for explanation of the individual species of catenanes.

(C) Quantification of scCMs from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).

(D) Quantification of Cats from (B) lanes 16-30. Mean ± SD, n=3 independent experiments. Abundance of catenanes was calculated as a percentage of total lane signal (% of total).

(E) Plasmid DNA was replicated in Mock- or TOP2α-immunodepleted extracts. At 60 minutes, undepleted extract was added back to add to supply TOP2α and trigger decatenation. Vehicle, etoposide, or doxorubicin were also added at 60 minutes. Samples were treated either with or without Proteinase K (as in Fig 2E).

(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. Linear molecules are indicated with a red arrow.

(G) Quantification of scCMs from + ProK gel in (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).

(H) Quantification of DPC Containing Molecules from (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of DPC-containing molecules was calculated as a percentage of total lane signal (% of total).
Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms (2022)
Figure 4

Figure 4. Etoposide stalls replication forks by trapping TOP2α behind forks

(A) Plasmid DNA was replicated in the presence or absence of etoposide and p97-i, which inhibits removal of replisomes (green) from DNA. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns.

(B) Proteins from (A) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. Modified TOP2α species are also indicated.

(C) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 1-8.

(D) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 9-16.

(E) Quantification of CDC45 from (B) lanes 1-8.

(F) Quantification of CDC45 from (B) lanes 9-16.

(G) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin (Aph), which prevents DNA synthesis but still permits unwinding by replisomes. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns.

(H) Proteins from (G) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction.

(I) Quantification of RPA30 from (H) lanes 9-16.

(J) Quantification of RPA30 from (H) lanes 1-8.

(K) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 1-4 and 9-12.

(L) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 5-8 and 13-16.

Data information: (C-F, I-L) Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment. See also Fig EV4B-F.
Adaptively evolved human oral actinomyces-sourced defensins show therapeutic potential (2021)
Figure 3

Figure 3. Antibacterial activity of AMSIN and its synergy with HNP1&2 or lysozyme C.

A Lysis of SSAN by AMSIN. Vacomycin was used as a control.

B Inhibition zone-based assay for synergy between AMSIN and HNP1&2 or lysozyme C (abbreviated as hLZc) to the two oral Streptococcus (SSAN and SSAL). Inset (upper panel, boxed), a representative example of the inhibition-zone test showing the synergy. Peptide doses used here were 0.05 nmol/well for HNP1&2 and hLZc at which no visible inhibition zones were observed for both of them. At the same dose, AMSIN produced a small inhibition zone. For synergetic observation, two peptides were applied to the same well according to the doses indicated. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test (***P<0.001; exact P values are listed in Table EV7). Note: the nanomolar concentration of HNP1&2 was calculated from the average molecular weight of HNP1 and HNP2.

C The FIC index assay for synergy between AMSIN and HNP1&2 or hLZc to SSAL and SSAN. The dashed line represents the cutoff of the FIC index (0.5) for synergy.

Source data are available online for this figure.
Inhibition of transcription by dactinomycin reveals a new characteristic of immunogenic cell stress (2020)
Figure 4

Figure 4. DACT-treated cells activate the immune system.

(A, B) Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry (A). The percentage of CMTMR and CD11c double positive cells among all CD11c+ cells are indicated (B). The mean of three independent experiments ± SEM is depicted, and p-values were calculated using pairwise multiple comparisons test with a Benjamin-Hochberg correction. Stars indicate significant differences between each treatment and its corresponding control at the same temperature, whereas hashes indicate significant differences comparing the co-culture at 37 °C to the co-culture at 4 °C for the same treatment: **/## p<0.01.

(A, C, D) MCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. Cells were collected and stained with a marker of viability, as well as with CD11c, MHCII and CD86 specific conjugated antibodies and analyzed by flow cytometry (A). The increase in the percentages of MHCII+ (C) and CD86+ (D) cells among CD11c+ cells was quantified with respect to untreated controls. The mean of five independent experiments ± SEM is depicted, and the p-values were calculated using a Student's t-test: * p<0.05, ** p<0.01, *** p<0.001 .

(E-G) 1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Dying cells were harvested and injected subcutaneously (s.c.) into one flank of immunocompetent syngeneic C57Bl/6 mice (n=8 mice) to assess vaccination efficacy. PBS was injected in the control group (Ctr). Two weeks later, animals were rechallenged with 1x105 untreated MCA205 cells in the contralateral flank of the animals (E). Next, tumor size was measured regularly and individual tumor growths of DACT-vaccinated versus Ctr mice are depicted (F). Overall survival is depicted and p-values (*** p<0.001.) were calculated with a Log-Rank test (G). One representative experiment among three is shown (F, G).