EMBO/soda-nel huggingface dataset:
Safe eradication of large established tumors using neovasculature-targeted TNF-based therapies (2019)
Figure 2

Figure 2. AFRs mediate target-specific delivery of TNF activity. (A) Schematic representation of AFR, consisting of N-terminal VHH fused via a 20xGGS linker to an engineered TNF (e-TNF) variant and C-terminal affinity tag. (B,C) Viability of L929 parental or mCD20-expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH (B) and viability of MCF7 parental or hCD20-expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH (C). Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM. (D) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus, measured via ELISA. Error bars are SEM. ns, non-significant; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test.
Safe eradication of large established tumors using neovasculature-targeted TNF-based therapies (2019)
Figure 3

Figure 3. Sc mTNF Y86F is non-toxic and mCD13-AFR is active in mice. (A) Toxicity of wt sc mTNF and mutant Y86F after injection (↑) in naïve C56BL/6 mice. First injection was i.v., other injections were i.p. Mean rectal body temperature ± SEM and cumulative survival rates are shown (n = 4). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C. (B) Plasma IL-6 concentrations measured via ELISA. Blood samples were taken 6 hours after i.v. injection of wt sc mTNF or mutant Y86F. **, p < 0.01; ns, non-significant by one-way ANOVA with Bonferroni's multiple comparison test. (C) Immunohistochemical staining for PECAM-1 (red), ICAM-1 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 7µg wt mTNF or 50µg of mCD13-AFR. Scale bar is 50µm. (D) Expression of ICAM-1, E-selectin and VEGF-R2 in B16Bl6 tumor 6h after treatment, by qPCR analysis of whole tumor RNA. Error bars are SEM. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test. (E) Tumor growth and body weight change after daily p.l. injection of indicated (equimolar) doses of mCD13-AFR, non-targeted AFR (BcII10-AFR) or mCD13 VHH control (CD13-hIFNα2) in the B16Bl6 melanoma model. The line under the graph represents the treatment period. Tumor growth is shown as mean TSI, error bars are SEM (n = 5). ***, p < 0.001 by two-way ANOVA with Bonferroni's multiple comparison test. (F) B16Bl6 tumor growth after daily p.l. treatment with indicated doses of mCD13-AFR, Wortmannin (Wm), Birinapant (Bir) or combinations thereof. Wortmannin and Birinapant were given 1 hour before CD13-AFR. Tumor growth is shown as mean TSI + SEM (n = 4 for Wm and Bir, 5 for other groups). The line under the graph represents the treatment period. (F) TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation. Error bars are SEM. ns, non-significant; *, p < 0.05; *** < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test.
Safe eradication of large established tumors using neovasculature-targeted TNF-based therapies (2019)
Figure 4

Figure 4. mCD13-AFR synergizes with immunotherapy. (A) Schematic representation of the CAR construct used, consisting of N-terminal anti-hCD70 VHH, hCD8α hinge and transmembrane (TM) domain, CD137 co-stimulatory domain and C-terminal CD3ζ activation domain. (B,C) SKOV3 tumor growth in NSG mice after i.v. injection of 6 x 106 hCD70 CAR T-cells on day 12 and p.l. treatment with 50µg mCD13-AFR (↑). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (B) or as TSI of individual mice (C). ***, p < 0.001 by two-way ANOVA with Bonferroni's multiple comparison test. (D) Flow cytometric analysis of hCD45+hCD3+eGFP+ CAR T-cells in SKOV3 tumors on day 18 after tumor inoculation. Mice were injected with 6 x 106 CAR T-cells on day 12 and treated with PBS or CD13-AFR (↑ in panel B). Two replicate tumors are shown. See Figure EV1A for gating strategy. (E) B16Bl6 tumor growth and body weight change after daily p.l. injection of 30µg mCD8-AFN, 50µg mCD13-AFR or a combination thereof. Tumor growth is shown as mean TSI + SEM (n = 6). The line under the graph represents the treatment period. (F) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation. Error bars are SEM. ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test.
Safe eradication of large established tumors using neovasculature-targeted TNF-based therapies (2019)
Figure 5

Figure 5. IFN-γ sensitizes tumor endothelial cells for TNF. (A,B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF, 30µg hTNF alone or in combination with 10000 IU mIFN-γ in wt C57BL/6 mice (A) or homozygous Flk1 dnIFN-γR1 transgenic mice (B). The line under the graph represents the treatment period. Tumor growth is depicted as mean TSI, error bars are SEM (n = 5). ns, non-significant; **, p < 0.01 by one-way ANOVA with Bonferroni's multiple comparison test. (C) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F (Y87F), hCD13-AFR or wt hTNF, alone or in combination with 200ng/ml hIFN-γ, measured via ELISA. Error bars are SEM. ns, non-significant; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test. (D) Schematic representation of AFN-II, consisting of a VHH fused via a 20xGGS linker to an engineered IFN-γ (e-IFN-γ) variant and N-terminal affinity tag. (E) Viability of B16Bl6 parental or mCD20-expressing cells after 72h stimulation with wt mIFN-γ or del8 mutant fused to a BcII10 or mCD20 VHH, in the presence of 60ng/ml mTNF. Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM.
Safe eradication of large established tumors using neovasculature-targeted TNF-based therapies (2019)
Figure 6

Figure 6. CD13-AFN-II synergizes with CD13-AFR for tumor destruction. (A,B) Tumor growth, body weight change and rectal body temperature after daily p.l. treatment with mCD13-AFR, mCD13-AFN-II or combination thereof, in the B16Bl6 model in C57BL/6 mice (A) or RL model in NSG mice (B). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (upper panel), or as TSI of individual mice (lower panel). The line under the graph represents the treatment period. (C) Immunohistochemical staining for PECAM-1 (red), cleaved Caspase-3 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 50µg mCD13-AFR and 23.8µg mCD13-AFN-II. Scale bar is 100µm.

Expanded View Figure Legends
MicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback (2021)
Figure 1

Figure 1. Lineage segregation of Hoxa5/Hoxc8-expressing cells in the developing spinal cord. (A) Expression patterns of Hoxa5 and Hoxc8 in MNs (demarcated by white dashed lines) along the rostrocaudal axis of the spinal cord (cervical C4 to thoracic T1) from mouse embryos from E9.5 to E12.5. Pink scale bars: 12.5 μm for E9.5; 12.5 μm for E10.5; 25 μm for E11.5; and 50 μm for E12.5.

(B) Quantification of Hoxon cells across MN domains. Data represent mean ± SD from N ≥ 3 embryos.

(C) Summary of the intrasegmental expression profiles of Hoxa5 and Hoxc8 in cervical MNs. Yellow squares represent the Hoxa5onHoxc8 on cells.
MicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback (2021)
Figure 3

Figure 3. Single-cell RNAseq and lineage tracing indicate coexistence of Hoxa5/Hoxc8 mRNAs in MNs along the spinal cord.

(A) Expression patterns of Hoxa5 and Hoxc8 mRNAs, as revealed by in situ hybridization, aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs (demarcated with pink/white dashed lines) along the cervical spinal cord (cervical C5 to C7) in mouse embryos at E12.5. Pink scale bar, 50 μm. White and pink dash lines demarcate the postmitotic MN region.

(B) Schematic illustration of the strategy to apply single-cell RNA sequencing (scRNAseq) on FACS-collected homogenous Hb9:GFPon MNs.

(C) Visualization of E12.5 cervical MNs (right panel) and the subset of LMCs (left panel) using Uniform Manifold Approximation and Projection (UMAP), a 2D non-linear transformation of high-dimensional data that assigns proximal x-y coordinates to cells (points) with similar expression profiles. Individual cells are colored according to their cluster assignments. Details are provided in the Materials & Methods.

(D) Dot-plots showing expression of genes (rows) that distinguish MN subtypes (columns). The height of each colored shape is proportional to the percentage of cells expressing the marker (≥1 UMI), and its width reflects the average transcript count within expressing cells.

(E) scRNAseq results reflect that Hoxa5/Hoxc8 mRNAs largely co-exist along the RC axis of E12.5 spinal cord, particularly at the middle boundary region.

(F) Schematic illustration of Hoxc8 lineage tracing. Hoxc8-expressing cells and all of their progeny are indelibly marked by tdTomato (RFP) expression in Hoxc8:Cre;ROSA26-loxp-STOP-loxp-tdTomato (lsl-tdTomato) embryos.

(G) Analysis of the Hoxc8 lineage (RFPon cells) in Hoxc8:Cre;ROSA26-lsl-tdTomato E12.5 spinal cord sections (cervical segments 5-6). RFP is expressed in a broad domain that includes Hoxc8on cells and a subset of Hoxa5on cells (Hoxa5on plus RFPon, indicated by arrowheads). Pink scale bar, 50 μm. White dash lines demarcate the postmitotic MN region.

(H) Hoxa5on and Hoxc8on cells adopt an "all or none" segregated cell fate despite coexistence of Hoxa5/Hoxc8 mRNAs, indicating a potential post-transcriptional regulatory mechanism for cell fate segregation.
MicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback (2021)
Figure 6

Figure 6. miRNA confers hysteresis in MN differentiation.

(A, B) Schematic illustrations of the experiments in (C) and (D). Embryoid bodies were incubated with: (i) RAhi (1 µM) for 48 h; (ii) RAhi for 72 h; (iii) RAhi for 48 h then switched to RAlow (100 nM) for 24 h; (iv) RAhi for 48 h then switched to RAlow plus RAR inhibitor (1 µM) for a further 24 h; (v) RAlow for 48 h; (vi) RAlow for 72 h.

(C, D) Immunostaining (C) of EBs 96 h after ESC differentiation, and quantification (D) of Hoxa5on in MNs (Hb9on).

(E) Schematic illustration of the experiments in (F) and (G). Inducible ESC lines expressing eight repetitive mir-27b sponge sequences were inserted into the GFP 3' UTR. ESCs were differentiated by doxycycline treatment after 48 h of differentiation. A scrambled sequence was inserted as a control.

(F, G) Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h.

(H) Bifurcation analysis of a mathematical model based on our mmi-3 Model. Steady states of the system in the presence of miR-27 (red, gray, and blue) or in the absence of miR-27 (purple) at various concentrations of RA are shown. Solid curve: stable steady state. Dashed curve: unstable steady state (See Appendix Supplementary Methods for parameter values).

Data information: Scale bar in (C) and (F) represents 50 μm. Data in (D) represent mean ± SD, N≥3 EBs from three independent experiments. The difference between each pair of groups with the same color is not significant (p > 0.05); the difference between each pair of groups with different colors (dark red vs. pink) show significant difference (p < 0.01), Student's t-tests. Scale bar in (F) represents 50 μm. Data in (G) represents mean ± SD, N≥3 EBs from three independent experiments, *p < 0.01, Student's t-tests.
MicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback (2021)
Figure 8

Figure 8. miR-27 and miR-196 mutants manifest a switched Hox boundary in MNs.

(A, B) Schematic illustration of the strategy to generate miR-27 and miR-196 knockout embryos.

(C-F) (C) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6/7 segments of miR-23~27~24 double KO (DKO) mice. miR-23a~27a~24-2+/-;miR-23b~27b~24-1-/- mice were used as a control (ctrl). (E) Conversely, Hoxc8 is shifted into the MNs of cervical C5 segments of both miR-196a2/b DKO and miR-196a1/a2/b triple knockout (TKO) embryos. Age-matched wild type embryos were used as controls. Scale bar represents 50 μm. White dash lines demarcate the postmitotoic MN domain (D, F) Quantifications of Hoxa5on, Hoxc8on, and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos (mean ± SD, N=3 embryos, *p < 0.01, Student's t-tests ).

(G) Summary of the Hox phenotypes in the miRNA mutants. Yellow squares represent the Hoxa5onHoxc8 on cells.
Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance (2022)
Figure 1

Figure 1. Mitochondria from REL neuroblastomas have attenuated apoptotic responses.

A Tumor models were derived at DX and REL following treatment. Mitochondria-rich fractions were exposed to tBid or BimBH3 peptide and cytochrome c release measured as a surrogate for apoptotic commitment.

B Maximal cytochrome c release in response to tBid or BimBH3 peptide for each replicate of a DX/REL tumor pair (1-9 biological replicates per DX/REL pair; n=44 total).

C Maximal cytochrome c release for all DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values).

D Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient-matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane).

Statistical analyses in C were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10). Cyto c, cytochrome c; mito, mitochondria.
Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance (2022)
Figure 2

Figure 2. REL neuroblastomas are resistant to multimodal cancer therapeutics.

A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136).

B DNA-damage induced by 2 Gy ionizing radiation as measured by γ-H2AX foci at 1hr (n=53-77 cell nuclei/condition scored; mean shown); statistical analyses performed using two-tailed Mann-Whitney U test.

C, D, E In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days, 48hr exposure to the Bcl2/Bclx-inhibitor, ABT-737, or 120hr exposure to the Alk inhibitor, crizotinib.

F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR).

G In vitro viability of NB1643 and NB1643-ALKR cells following 72hr exposure to ABT-737.

Data information: For A, C-E, and G data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted-line represents 50% viability. For F, data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.
Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance (2022)
Figure 4

Figure 4. Chemically or genetically reducing MAMs leads to MOMP resistance.

A Heavy membrane (HM) fractions of CHLA15 were rendered MAM-depleted by immunomagnetic separation to derive purified mitochondria (pMito), and cytochrome c release measured in response to BimBH3 peptide.

B, C, D TEM analyses were used to quantify mitochondrial size, circularity and roundness in CHLA15 cells transfected with a sh-control (Ctrl), shMFN2 or shPACS2 constructs (mean +/- SD shown).

E Immunoblot assessment of Mfn2 and Pacs2 protein knockdown.

F Proportion of mitochondria with 0 to 5 MAMs are shown for each.

G Percentage of mitochondria perimeter with an apposed ER within defined gap-widths; dotted-lines denote +10% and -10% change.

H In vitro viability of CHLA15-ctrl, CHLA15-shMFN2, CHLA15-shPACS2, and CHLA20 cells following 72hr exposure to ABT-737; dotted-line represents 50% viability.

I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15-Ctrl, CHLA15-shMFN2 and CHLA15-shPACS2 cells.

Data information: For B-D and F-G, n=214-246 mitochondria per cell line. For A and I, data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates; for H, data points are mean and SD from triplicate wells, experiments are representative of at least two biological replicates. For B-D, statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05.
Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance (2022)
Figure 5

Figure 5. Reduced Ca2+ transfer at MAMs is not required for attenuated mitochondrial responses to stress.

A, B Cytosolic (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an IP3R-agonist; time added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time between achieving 50% of maximal cytosolic and mitochondrial concentrations) is an index of ER-mitochondrial proximity and transfer efficiency; plotted below.

C MAM proximity was enforced in SKNBE2C cells using an ER-OMM linker construct, with Ca2+ transfer coupling time (n=57 cells) compared with cells expressing an OMM-only control linker (n=70 cells); tracings are mean +/- SEM error bars.

D Mitochondrial cytochrome c release for CHLA15 and CHLA20 cells following exposure to tBid or BimBH3 peptide, with or without Ru360 treatment.

E Silencing of the mitochondrial calcium uniporter (MCU) was achieved in two CHLA15-shMCU clones.

F Cytochrome c release in response to tBid and BimBH3 assessed.

Data information: For A-C, data is derived from at least 3 separate cell transfections (biological replicates). For D and F, data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates. For A-C, statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05.
Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence (2020)
Figure 1

Figure 1 A screen for telomere associated proteins in yeast

A) Schematic representation of the quantitative interactomics approach to identify proteins binding to the S. cerevisiae telomeric sequence.

B) Volcano plot of quantified proteins. Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p-values were calculated with a Welch t-test. Proteins above the threshold p=0.05, s0=1, c=0.5 (dashed line) are considered enriched.

C) Gene ontology enrichment analysis of the telomere-sequence associated interactors. Analysis was performed using the PantherDB.org overrepresentation Test. Fisher's exact test was subjected to Bonferroni correction.

D) Heatmap of LFQ intensity of proteins enriched at DNA baits. Telomere interactors are indicated on the right side of the heatmap. Color code indicates measured log2 LFQ intensities in the telomere pull-down. RNA binding proteins are marked with a black box on the left side of the figure.

E) Critically short telomeres arise after 90 population doublings in the absence of telomerase (tlc1). Genomic DNA was extracted at indicated population doublings and digested with XhoI. A radioactive probe was used to recognize telomeric DNA in a southern blot.

F) tlc1 cells reach senescence at PD 80-100. %viability of indicated strains after propagation in YPD is indicated at different population doublings n=3. Asterisk indicates population doubling used in G) to determine binding of Npl3 to short telomeres by TAP-ChIP. 3 independent spores from the indicated genotypes were used. Population doublings are counted from germination of meiotic products and hence begin at approx. PD40.

G) Npl3-TAP associates strongly to short telomeres. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). PD90 refers to 90 population doublings in the absence of TLC1 (telomerase RNA subunit). The no tag control serves as an indicator of non-specific background signal.

Abbreviations: LFQ: Label free quantification, MS: mass spectrometry, RBP: RNA binding protein, TAP: tandem affinity purification tag, PD: population doubling.
Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence (2020)
Figure 2

Figure 2 Npl3 associates to telomeres in a TERRA-dependent manner

A) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A, B and using tlc1 protein extracts. Interactors were identified using untreated protein extracts from tlc1 cells (left panel) or digested with RNase A and RNase H (right panel).

B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01).

C-E) Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01).

F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01).

H) Npl3 associates with TERRA at telomeres, likely through association with the nascent transcript. When Rat1 is absent, TERRA is stabilized and Npl3 would persist at telomeres bound to TERRA. RNaseH1 resolves telomeric RNA-DNA hybrids, which may release nascent TERRA with Npl3 bound.

Abbreviations: eV: empty vector control, oE: overexpression, IAA: indole-3-acetic acid, AID: Auxin inducible degron. Individual p-values are indicates as "p=".