EMBO/soda-nel huggingface dataset:
Functional Skewing of TRIM21-SIRT5 Interplay Dictates IL-1β Production in DSS-induced colitis (2022)
Figure 7

Figure 7. Mutual regulation of SIRT5 and TRIM21 influences IL-1β production in LPS-activated macrophages and DSS-induced colitis in mice.

(A, B) SIRT5-/- macrophages (BMDMs) were transfected with HA-SIRT5 WT or mutants in the absence or presence of Flag-TRIM21 as indicated. Macrophages were stimulation with 100 ng/mL LPS for 4 hours. mRNA expression of IFN-β (A) and IL-1β (B) was determined by qRT-PCR analysis.

(C, D) TRIM21-/- macrophages (BMDMs) were transfected with Flag-TRIM21 WT or mutants in the absence or presence of HA-SIRT5 as indicated. Macrophages were stimulation with 100 ng/mL LPS for 4 hours. mRNA expression of IFN-β (C) and IL-1β (D) was determined by qRT-PCR analysis.

(E) Genotyping of TRIM21-/-, SIRT5-/- and TRIM21-/-SIRT5-/- mice. DNA derived from the tail of wild type and knockout animals was analyzed by PCR. Protein expression of TRIM21 and SIRT5 in BMDMs derived from mice were also analyzed by western blotting.

(F-I) wild-type (WT, n=5), TRIM21-/- (n=5), SIRT5-/-(n=5), and TRIM21-/-SIRT5-/- (n=5) mice were orally administrated with 2.5% (w/v) DSS in drinking water for 7 days and regular drinking water for another 2 days. Mice body weight (F) and Colon-length (G) changes were measured. The colon tissues were examined microscopically for pathological changes (H, left panel, scale bar: 100μm), which were further semiquantitatively scored (H, right panel). (I) IL-1β mRNA expression in colon tissues determined by qRT-PCR.

Data information: Data are means ± SD. In A-D, n=3 biological replicates, in F-I, n=5 biological replicates. Western blots in E represent three independent experiments. P values were determined by unpaired two-tailed Student's t-tests. ns, not significant; ***p < 0.001, ****p < 0.0001.

Expanded View Figure legends
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization (2006)
figf1

(a) Cartoon of a single cell before and after trypsin treatment showing the localization and topology of YFP-PrP (green) and HLA-A2–CFP (red) on the plasma membrane. (b) N2a cells coexpressing YFP-PrP and HLA-A2-CFP were subjected to 6 mM trypsin for 100 s. Images were taken before and after trypsin treatment as indicated. Note that the membrane signal of YFP-PrP was destroyed by the protease, whereas HLA-A2–CFP's membrane signal remained unaffected. (c) Cartoon of a single cell before and after digitonin treatment. The green balls represent GFP and its localization in the cytosol and nucleoplasm. (d) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmic GFP. N2a cells expressing GFP were treated with 20 μM digitonin. Images were taken before and after digitonin application at the indicated time points. Scale bars, 10 μm.
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization (2006)
figf2

(a) Cartoon of the FPP assay illustrating a single cell before and after digitonin and trypsin treatment. The red balls represent RFP and its localization in the cytosol and nucleoplasm. The green balls represent CD3δ-CFP in the ER and the position of the fluorescent protein tag relative to its respective membrane. (b) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ-CFP. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization (2006)
figf3

(a) Cartoon of the FPP assay illustrating the cellular localization of ER-bound CD3δ-CFP (green) and YFP-CD3δ (red), and the position of the fluorescent protein tag relative to its respective membrane. (b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm. (c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization (2006)
figf4

(a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing GalT-YFP and CD3δ-CFP were subjected to the FPP assay, as in Figure 2b. (c) Cartoon illustrating localization of mitochondrial EYFP-Mito (red) and Cd3δ-CFP (green). (d) N2a cells coexpressing EYFP-Mito and CD3δ-CFP were subjected to the FPP assay, as in b. Scale bars, 10 μm.
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization (2006)
figf5

(a) Cartoon of the FPP assay illustrating the cellular localization of RFP-SKL (red) and Pex3p-GFP (green) and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing RFP-SKL and Pex3p-GFP were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin as indicated. Scale bar, 10 μm.
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization (2006)
figf6

(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.
XIAP represents a promising therapeutic target for relapsed/refractory ALL (2022)
Figure 1

Figure 1: Smac mimetics sensitize r/r ALL towards cytotoxic drugs

A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine (VCR) for 48h. Apoptosis was determined by flow cytometry upon staining with Annexin-V and Propidium Iodide (PI). Mean ± SD of 3 independent experiments is shown; ** p <0.01, *** p <0.001 and **** p <0.0001 by unpaired Student's t-test.

B Fresh primary leukemic cells from two pediatric patients, one with common B-ALL at relapse (left) and one with T-ALL at diagnosis (right), both with poor response to second line chemotherapy, were treated with LBW242 and VCR at the indicated concentrations. Apoptosis was measured after 24 h as described in A.

C-F The SM-drug combination activates caspases and induces PARP cleavage. LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with respective fluorescent substrates for caspase-8 (C) and -9 (D) or an antibody detecting the active caspase-3 subunit (E), and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test. Cleavage of PARP was measured by Western blot using Actin as loading control (F). One representative immunoblot out 3 independent experiments is shown.

Source data are available online.
XIAP represents a promising therapeutic target for relapsed/refractory ALL (2022)
Figure 2

Figure 2: Apoptosis induction is independent from TNFα, RIPK1 and NF-κB

A Necrostatin-1 (Nec) does not affect LBW-mediated sensitization to VCR-induced apoptosis. Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and/or VCR (left) and/or TNFα (right) in the presence or absence of the RIPK1-inhibitor Nec. Mean ± SD of 3 independent experiments is shown; * p <0.05 and ** p<0.01 by unpaired Student's t-test.

B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR. LOUCY cells were transfected with a non-targeting siRNA (neg-siRNA) or a RIPK1-specific siRNA. 6h post transfection, cells were treated with the indicated drugs and apoptosis was measured 40h thereafter. Mean ± SD of 3 independent experiments is shown; *** p<0.001 by unpaired Student's t-test. Knockdown (KD) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control. One representative immunoblot out of 3 biological replicates is shown.

C The SM-drug combination does not activate the canonical NF-κB pathway. Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and/or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5, 15 or 60 minutes using Actin as loading control. One representative immunoblot out of 3 independent biological replicates is shown.

D KD of NEMO does not affect the SM-mediated sensitization to chemotherapy-induced apoptosis. Experiment was performed as described in B, except that an siRNA targeting NEMO was used. Mean ± SD of 3 independent experiments is shown; * p<0.05 by unpaired Student's t-test.

E The SM-drug combination activates the non-canonical NF-κB pathway. Cleavage of p100 NF-κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM. Asterisk indicates an unspecific signal. One representative immunoblot out of 3 biological replicates is shown.

F KD of RelB does not inhibit apoptosis. Experiment was performed as described in B, except that an siRNA targeting RelB was used. Mean ± SD of 3 independent experiments is shown; differences are not significant, by unpaired Student's t-test.

Source data are available online.
XIAP represents a promising therapeutic target for relapsed/refractory ALL (2022)
Figure 3

Figure 3: XIAP is upregulated in primary ALL patient samples and overexpression regulates cell death induction

A Expression of XIAP mRNA in samples obtained from BM of healthy individuals (Ctrl, n=10) or patients with AML (n=517) or ALL (n=306). Data are expressed as median, 25th and 75th percentile, whiskers indicate min/max; ** p<0.01; **** p<0.0001 by Games-Howell post-hoc test.

B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients, analyzed by protein immunoassay (Simple Western) and depicted as mean (n=2, biological replicates); PBMC = peripheral blood mononuclear cells (see Appendix Figure S2B).

C,D Parental and XIAP over-expressing NALM-6 cells (C) or REH cells (D) were treated with VCR or LBW242; apoptosis (Annexin-V / PI) was measured at the indicated time points (C) or after 72h (D) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed in parental and XIAP-overexpressing NALM-6 cells by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.

Source data are available online.
XIAP represents a promising therapeutic target for relapsed/refractory ALL (2022)
Figure 5

Figure 5: XIAP plays an essential role for patients' r/r ALL cells growing in mice

A Generation of ALL genetically-engineered PDX (GEPDX) models. Primary patients´ALL-265 cells were transplanted into mice to generate serially transplantable PDX cells. PDX cells were lentivirally transduced in two rounds with the constructs depicted in Figure 4a, and fluorochrome+ PDX cells were enriched by flow cytometric cell sorting; resulting cells expressing either sh-CTRL/mTagBFP or sh-XIAP/eGFP were amplified and applied for repetitive in vivo transplantation assays.

B-C XIAP plays an essential role for ALL-265 growing in mice. Experimental scheme and stability of XIAP knockdown over passaging. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). XIAP expression levels of P1 and P2 PDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were analyzed by Western blot at the end of each passage; GAPDH was used as loading control (B). Relative proportion of ALL-265 GEPDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP re-isolated from the BM at the end of each passage was quantified by flow cytometry (C). Data are depicted as mean ± SEM of all mice analyzed (n=4, each passage) with **** p<0.0001 by unpaired Student's t-test.

D Dose-response relationship between strength of XIAP knockdown and growth disadvantage; ALL-265 PDX cells of a representative mouse were analyzed separately for dsRED/shRNA high and low expressing cells.

Source data are available online.

Expanded View Figure Legends
OXPHOS deficiency activates global adaptation pathways to maintain mitochondrial membrane potential (2021)
Figure 3

Figure 3. Transcriptional repression of Inh1 maintains ∆ψm, ISC synthesis, and proliferation of ρ0 cells.

qRT-PCR analysis of INH1 mRNA levels. Data are mean ± SD from three biological replicates.

Western blot analysis of Inh1 protein levels. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.

Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ and 37 ℃ for 2 days.

Western blot analysis of copper-inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours.

Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis.

Overexpression of Inh1 represses the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30℃ for 2 days.

Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC-containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Nfs1, Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA.

Cartoon illustration of the translational repression of Hap4 and the subsequent transcriptional downregulation of Inh1 to maintain ∆ψm in ρ0 cells.

The red ] highlight two cell lines for comparison (E, F).
OXPHOS deficiency activates global adaptation pathways to maintain mitochondrial membrane potential (2021)
Figure 6

Figure 6. Snf1/AMPK upregulates glycolysis and represses ribosome biogenesis to maintain ∆ψm.

Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.

Snf1 represses ribosome biogenesis in WT and ρ0 cells. Polysomes of the indicated strains were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.

Deletion of SNF1 decreases ∆ψm in both ∆V and ρ0 cells.

Deletion of SNF1 decreases the proliferation of both ∆V and ρ0 cells. Serial dilutions of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.

Deletion of MIG1 in ρ0 snf1∆ cells rescues ∆ψm.

Deletion of MIG1 in ρ0 snf1∆ cells partially rescues cell proliferation. A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.

Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1∆ cells fully rescues cell proliferation. A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.

Cartoon illustration that Snf1 signaling in ∆V and ρ0 cells upregulates glycolysis and represses ribosome biogenesis to optimize energy metabolism.

Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis (C, E). The red ] highlight two cell lines for comparison (C, D, E).
OXPHOS deficiency activates global adaptation pathways to maintain mitochondrial membrane potential (2021)
Figure 7

Figure 7. Upregulation of chaperones maintains ∆ψm.

Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red.

Summary of the growth results of the indicated strains. -: no effect on growth. +: growth inhibition. ++: strong growth inhibition. Genes that are preferentially required in ρ0 puf3∆ cells are highlighted in red.

Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3∆ cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Data are mean ± SD from three biological replicates.

Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.

Cartoon illustration of the progressive activation of adaptive responses to convergently maintain ∆ψm in OXPHOS mutants.

Expanded View Figure legends
Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles. (2022)
Figure 2

Figure 2. Optimisation of production and physical characterisation of TPA.

(A) Yield comparison from standardised particle production using either superinfection by RGD4C.M13KO7 of E. coli transformed with TPA plasmid (i), or a calcium chloride competent E. coli carrying the RGD4C.M13KO7 genome (c), with or without kanamycin for selective expression, and at different incubation time endpoints. Data are expressed as mean + SEM, and experiments were repeated three times (n = 3).

(B) Percentage yield comparison between optimised TPA and AAVP production protocols, normalised to the TPA yield. Data are expressed as mean + SEM from three independent experiments (n = 3), one-way ANOVA with Tukey HSD test was used.

(C) Comparison of mean TPA and AAVP particle length, using a TEM. Scale bar, 200 nm. Independent t-test was used for analysis of data shown in the graph, n = 5, technical replicates.

(D) SmaI digestion of plasmid DNA extracted from E. coli at 18 hours of TPA.GFP particle production. The TPA.IRES.GFP DNA extract was used as positive control to confirm the separation of the two bands and thus complete digestion. Undigested TPA DNA was included and BamHI enzyme digestion was used to linearise the TPA plasmid. Extracts from 18 hours outgrowth of bacteria carrying either TPA.GFP or helper phage were also digested.

(E) Quantification of relative intracellular TPA DNA accumulation in HEK293 cells 2 hours post-transduction with either targeted (RGD4C) or non-targeted (NT) TPA, 0.5x106 TU/cell, using qPCR and annealing primers specific to the ITR sequence from AAV-2. Data are expressed as mean + SEM. The experiment was repeated twice in triplicate and the results shown are representative of one experiment. Independent t-test was used for data analysis.

(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.
Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles. (2022)
Figure 4

Figure 4. Targeted TNFαIL2 cytokine treatment of solid tumors.

(A) TPA particle DNA carrying the sequence encoding a secreted human soluble TNFα, whose secretion is controlled by the human IL2 signal peptide (sp).

(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.

(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.

(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment.

(E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.
Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles. (2022)
Figure 5

Figure 5. Design, construction and in vitro investigation of TPA carrying IL15.

(A) TPA DNA carrying a sequence encoding a mouse IL15IgK whose secretion is under the mouse IgK sp.

(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis

(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.

(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.