EMBO/soda-nel huggingface dataset:
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 4

Figure 4. Long-range RMD is mediated by BIR.

A, Schematic drawing of the long-range RMD-GFP reporter in mES cells. Two 287 bp repeats are located separately in the Cdkn1A and Pim1 gene loci and are 0.4 Mbp apart. GFP+ cells would be generated after RMD.

B, Impact of the 5' DSB/repeat distance on RMD frequency when the 3' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.

C, Long-range RMD is dependent on POLD3 when one DSB is close to a repeat. mES cells carrying the RMD-GFP reporter were transfected with nontargeting siRNA (Ctrl) or a pool of POLD3 siRNAs (siPOLD3) 24 hours before for the first time, and with transfection of sgRNAs/Cas9 the second time. The graphs show the frequency of GFP+ cells normalized to the transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated. Depletion of POLD3 and RAD51 by transient transfection with siRNA was determined by qPCR, as shown in Appendix Fig S7A. Cells transfected with non-targeting control siRNA were used as control.

D and E, Impact of the 3' DSB/repeat distance on RMD frequency when the 5' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.

F, RMD frequency of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9, with the frequency of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.

G, The roles of POLD3, RAD51 and RAD52 in RMD of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9 after POLD3 (left) or RAD51 (middle) was depleted by siRNA or RAD52 was knocked out (right). The distance between the sgRNA targeting sites and the repeats is indicated.

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns, not signiﬁcant, two-tailed non-paired t-test.
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 5

Figure 5. The role of ATM and H2AX in RMD

A and B, SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells (A) and BIR/RMD in mES cells carrying the RMD-GFP reporter (B) were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 (A and B), or in ATM KO mES cells carrying the RMD-GFP reporter (right).

C and D, Dependence of RMD on H2AX. SSA/RMD and BIR/RMD frequencies in U2OS (EGFP-SSA-3427-17) cells (C) and BIR/RMD in mES cells carrying the RMD-GFP reporter (D) were determined using the indicated sgRNA/Cas9 after depletion of H2AX by lentivirus expressed shRNA (C), or by transfection with H2AX siRNA or in RAD52 KO cells (D). Depletion of H2AX by shRNA in (C) and by siRNA in (D) was determined by qPCR shown in Appendix Fig S8B and C, respectively.

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns, not signiﬁcant, two-tailed non-paired t-test.
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 6

Figure 6. Efficient long-range BIR/RMD requires DSBs that are present in the vicinity of both repeats.

A, Frequency of long-range RMD is increased by an additional DSB downstream of the 3' repeat. Illustrations show the GFP-RMD reporter, with sgRNAs/Cas9 cleavage sites indicated as red arrows (left). The frequency of BIR/SSA in mES cells carrying the RMD-GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs/Cas9 (right).

B and C, The effects of RAD52, POLD3, ATM and H2AX inactivation on BIR/RMD with cleavage at L17/D10k. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter was determined using the sgRNAs/Cas9 at L17/D10k in RAD52 KO cells or after POLD3 depletion by siRNA (B), or after treatment with ATM inhibitor (ku55933, 15mM) or H2AX depletion by siRNA (C).

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed non-paired t-test.
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 7

Figure 7. Long-range BIR/RMD is inhibited by divergence in repeat sequences.

A, Long-range BIR/RMD with identical repeats or divergent repeats. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter with identical repeats (0%) or repeats with 1% and 3% divergence was determined after cleavage at L17/R3.3k or L17/R28.4k.

B and C, The effect of MSH2 on BIR/RMD when repeat sequences are divergent. The frequency of BIR/RMD was determined after cleavage at L17/R3.3k or L17/R28.4k in mES cells carrying the RMD-GFP reporter with 1% and 3% divergent repeats, and with and without MSH2 KO (B) or with MSH2 KO and POLD3 depletion by siRNAs (C). The MSH2 KO was validated by Western blot, using KU70 as the loading control as shown in (B).

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed non-paired t-test.
HIV-1 infection of CD4 T cells impairs antigen-specific B cell function (2020)
Figure 1

FIG 1. NEF expression in CD4 T cells blocks their T helper function resulting in a lack of antibody production

(A) Outline of the experimental setup to assess the immunomodulatory effects of HIV-1 NEF on the generation of humoral immune responses. Antigen-specific interactions are established using HEL-OVA as model antigen with HEL-specific SWHEL B cells (BHEL) and OVA-specific OT-II CD4 T cells (CD4OT-II).

(B, C) Levels of anti-HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL-OVA/CFA following adoptive transfer of BHEL cells without CD4OT-II cells (no T cell control) or with control CD4OT-II cells or NEF-expressing CD4OT-II cells. (B) NEF WT of HIV-1SF2 (C) patient-derived NEF variants. Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1. Each dot represents one animal. Bars represent mean values with SD from 2-10 mice analyzed from 3 (B) and 2 (C) independent experiments. Statistical significance was assessed by Mann Whitney U test. ***P ≤0.001, *P ≤0.05
HIV-1 infection of CD4 T cells impairs antigen-specific B cell function (2020)
Figure 4

FIG 4. HIV-1 NEF prevents GC formation upon immunization

NEF impairs GC formation in immunized mice via the 12-39 region.

(A) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm.

(B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C.

(D-G) Flow cytometry dot plots of cells in dLNs from mice 7d post immunization that received the indicated transduced CD4OT-II and BHEL cells. Adoptively transferred cells were identified and distinguished from endogenous cells of recipient SMARTA mice (CD45.1+) by staining for CD45.2. For quantification of absolute cells numbers, precision beads were used and cells double positive for B220 and CD45.2 (D, BHEL cells) or CD3 and CD45.2 (F, CD4OT-II cells) analyzed. (E, G) Quantification of absolute BHEL cells (E) as shown in D and CD4OT-II cells (G) as shown in F. Shown are mean values with SD of 6 mice from 3 independent experiments. Each dot represents one animal.

Data information: Statistical significance was assessed by One-Way ANOVA test with Holm-Sidak's multiple comparison test. ***P ≤0.001, ** P≤0.01, *P ≤0.05
HIV-1 infection of CD4 T cells impairs antigen-specific B cell function (2020)
Figure 5

Fig 5. NEF restricts T-B cell interactions upon immunization

NEF impairs duration of Ag-induced interaction of CD4OT-II cells with BHEL cells in vivo.

(A) Still images of the Movies EV3 (Control, upper panel) and EV4 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel), respectively. High endothelial venules (HEVs) (white) were labeled by intravenous injection of Alexa660-labeled Meca-79 antibody and collagen (blue) is visualized by second harmonic generation (SHG). Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm.

(B) Quantification of the duration of interactions as indicated by the arrows in A. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Data is derived from 5 to 6 movies, acquired from 4 mice in 2 independent experiments. Percentages on top indicate frequency of interactions that lasted 20min or longer.

(C) Still images of the Movies EV5 (Control, upper panel) and EV6 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel). Follicular dendritic cells (FDCs) (white) were labeled by subcutaneous injection of Alexa647-labeled anti-CD21/CD35 antibody and collagen (blue) is visualized by SHG. Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm.

(D) Quantification of the duration of interactions as indicated by the arrows in C. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Shown is one representative dataset out of two, derived from 5 to 8 movies, acquired from 4 mice. Percentages on top indicate frequency of interactions that lasted 20min or longer.
Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages (2020)
Figure 2

Figure 2. Expression of H5N1 PB1-F2 Delays Pyroptotic Cell Death

(A-D) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Flow cytometry results for annexin V single positive (A and C) and annexin V and zombie dye double positive (B and D) cells. Percentage of positive cells are depicted at 12 hpi or 24hpi.

(E and F) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi (E) or 24hpi (F).

Data information: Mean values of n=3 independent experiments measured in technical duplicates are indicated (A, B and E) or n=3 independent experiments for (C and D) or n=4 independent experiments for F. Statistical analysis was performed by one-way ANOVA, p-values are indicated.

G THP-1 Caspase 1 -/- cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi. n=3 for 12hpi, n=5 for 24hpi. Medians are indicated, statistical analysis was performed by one-way ANOVA.
Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages (2020)
Figure 3

Figure 3. The PB1-F2 Null Mutation Amplifies NLRP3-dependent Inflammasome Activation in H5N1 Infected Cells

(A) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of the full-length gasdermin D. Equal loading was confirmed by beta actin. One representative blot of five independent repeats is shown.

(B) Quantification of band intensity for the gasdermin D full-length isoform. The mean ± standard deviation five independent experiments is shown. Statistical analysis was performed by the paired two‑tailed Student's t test., The asterisk (*) marks a significant difference when compared to the mock group ** p < 0.01.

(C) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of cleaved gasdermin D N-terminal and C-terminal isoforms by immunoblot in supernatants; one representative experiment of three independent repeats is shown.

(D) Quantification of band intensity for N-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05.

(E) Quantification of band intensity for C-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05.

(F) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Pro-caspase-1 and active caspase-1 were detected by immunoblot in cell lysates (cell, upper panels) and supernatants (sup, lower panels) from the same experiment. One representative experiment of three independent repeats is shown.

(G) Quantification of band intensity for active caspase-1 band. Five independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. * p < 0.05.

(H) Levels of total pro-caspase-1 were quantified on pellets and respective supernatants. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test.
Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages (2020)
Figure 4

Figure 4. PB1-F2 Interacted with NLRP3 in a Strain Dependent Manner

(A) Western blot analysis of whole cell lysate (input) and Flag-specific immunoprecipitation (IP) from 293T cells co-transfected with expression plasmids encoding V5-NLRP3 and Flag ZsGreen-PB1-F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown.

(B) Quantification of the NLRP3 IP band was normalized to the respective NLRP3 input band (n=3 independent biological replicates, normalized to the respective input). Mean values +- SD are indicated.

(C) Alignment of PB1-F2 sequence from two avian IAV strains (VN (H5N1) and Sha (H7N9)) and two mammalian/human IAV strains (PR8 (H1N1) and Wyo (H3N2)) (Combet et al., 2000). * above the sequence shows a conserved amino acid between the four strains while ↑ on the bottom shows a different amino acid for PR8 in comparison to the other strains.

(D) Schematic diagrams showing the truncated versions of Flag ZsGreen PB1 F2 used in Figure 6E (not to scale).

(E) 293T cells co transfected with expression plasmids encoding different truncation mutants for Flag Zsgreen-PB1-F2 from VN (H5N1) and V5-NLRP3 were processed as described in (A). Representative blot of three independent repeats is shown.
Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages (2020)
Figure 5

Figure 5. Mechanism for PB1-F2 Interaction with NLRP3 hence Preventing Nek7 Binding

(A) Schematic diagrams showing the NLRP3 constructs used in 7B.

(B) 293T cells were co-transfected with expression plasmids encoding for the indicated V5-tagged domains of NLRP3 for 24h. Whole cell lysate (left blot) and flag specific IP (right blot) were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of two independent experiments are shown.

(C) 293T cells were co‑transfected with expression plasmids encoding Flag-NLRP3, V5‑PB1-F2 and V5‑ASC for 24h. Whole cell lysate and flag specific IP were analyzed by immunoblotting for indicated antigens. Equal loading of whole cell lysate was assured by blotting against beta actin. Representative blot of two independent repeats is shown.

(D) 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and Flag IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. Right panel: Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05).

(E) As in D: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown.

(F) Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05).

(G) As in D and E: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or V5-NLRP3-Pyrin or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of three independent experiments are shown.

Expanded View Figures:
S1P Defects Cause a New Entity of Cataract, Alopecia, Oral Mucosal Disorder, and Psoriasis-like Syndrome (2022)
Figure 2

Figure 2. S1P dysfunction impairs mitochondrial import and results in mitochondrial abnormalities.

A, B Representative transmission electron microscopy (TEM) images showing keratinocyte mitochondria (red arrow) in patient 1 (A) and in the Mbtps1-conditional knockout (cKO) mouse model (B). Scale bars: 2 µm.

C, D Quantification of the mitochondrial number and morphology in patient 1 (C) and the Mbtps1-cKO mouse model (D). (C) Left panel: n = 13 biological replicates of normal individuals, n = 10 biological replicates of patient 1. Middle and right panel: n = 121 biological samples of normal individuals and patient 1. (D) Left panel: n = 23 biological replicates of Mbtps1-loxP mice; n = 15 biological replicates of Mbtps1-cKO mice. Middle panel and right panel: n =113 biological replicates of Mbtps1-loxP mice and Mbtps1-cKO.

E Immunofluorescence experiments showed that mutant S1P (p.Val355Gly and p.Ter1053Arg) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild-type S1P, which suggested that these variants disrupt its mitochondrial import. The white arrowheads indicate the colocation (yellow) of S1P (green) and the mitochondrial tracer (red). Scale bars: 20 μm (panels 1-4); scale bars: 2 μm (panel 5).

F Cellular component separation assay showing that S1P was enriched in mitochondria. Biochemical fractionation of the whole cell lysate (WCL), cytosol (Cyto, tubulin), mitochondria (Mito, COX IV), lysosomes (Lyso, LAMP1), Golgi apparatus (Golgi, GM130), endoplasmic reticulum (ER, ERp72), and nucleus (Nue, Lamin B) from HaCaT cells.

G A component separation assay revealed a lower expression of mutant S1P (p.Val355Gly and p.Ter1053Arg) than wild-type S1P in mitochondria.

H The impaired binding of mutant S1P (p.Val355Gly and p.Ter1053Arg) to translocase of the outer membrane (TOM) 70 and translocase of the inner membrane (TIM) 23 was detected by a coimmunoprecipitation (Co-IP) assay.

Data information: The data are presented as the means ± SDs. Statistical significance was assessed by the mean-Whitney two-tailed U test (C and D). ***P < 0.001. Three biological replicates were included in the study (E-H).
S1P Defects Cause a New Entity of Cataract, Alopecia, Oral Mucosal Disorder, and Psoriasis-like Syndrome (2022)
Figure 3

Figure 3. S1P forms a trimetric complex with electron transfer flavoprotein (ETF) A and ETFB proteins

A LC-MS/MS analysis of S1P-binding proteins. Total protein lysate was subjected to coimmunoprecipitation (Co-IP) with normal IgG or S1P antibody. The purified protein complex was separated by SDS-PAGE and then subjected to silver staining. The arrows indicate the bands containing S1P, ETFA and ETFB. The differential bands were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

B Role of the S1P-ETFA-ETFB interaction network in mitochondrial and nonmitochondrial protein systems.

C S1P interacts with endogenous ETFA and ETFB in HaCaT cells. Total protein lysate was subjected to Co-IP with normal IgG, ETFA (left panel) or ETFB (right panel) antibody. The interaction between S1P and ETFA/ETFB was then detected by immunoblotting.

D The direct interaction between S1P and ETFA-ETFB was validated by a GST pull-down assay. Left panel: in vitro-translated S1P was pulled down by purified GST-ETFA fusion protein. Right panel: in vitro-translated S1P was pulled down by purified GST-ETFB fusion protein.

E Confocal immunofluorescence demonstrated that S1P colocalized with ETFA and ETFB in HaCaT cells. The black arrowheads indicate the colocalization (white) of S1P (green), ETFA (red) and ETFB (purple). Scale bars: 20 μm (panels 1-5); scale bars: 2 μm (panel 6).

F Three-dimensional structure of the S1P-ETFA-ETFB-FAD complex in stereo. The structures of S1P (magenta), ETFA (light blue), ETFB (green) and FAD (red) are depicted in carbon.