EMBO/soda-nel huggingface dataset:
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 6

Figure 6. Fbxl17 mediated degradation of Sufu promotes medulloblastoma tumour growth

A. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with control shRNA or shRNA targeting Fbxl17. (mean ± SEM from 3 independent experiments, ***p<0.0005).

B. Detection of Sufu protein levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. Representative image of three independent experiments is shown.

C. Quantification of Gli1 mRNA levels in DAOY cells transfected with control shRNA or shRNA Fbxl17. (mean ± SEM from 3 independent experiments, **p<0.005).

D. T2- weighted magnetic resonance images showing tumour development in rats injected with DAOY cells transfected with control shRNA or shRNA against Fbxl17. One representative image for each condition, at 10 weeks is shown. Scale bar: 5mm. E. Graph showing area of T2 hyperintensity (a surrogate marker of tumour growth) between 4 and 10 weeks post-tumour induction. Animals were injected with DAOY cells stably expressing control shRNA or shRNA against Fbxl17 (10,000 cells/1µL). (mean ± SEM; n = 5; two-way ANOVA, followed by unpaired t-test, *p<0.05, **p<0.01).

F. Representative immunohistochemical image of Vimentin staining. Scale bar: 1 mm.

G. Quantification of tumour growth (vimentin staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).

H. Representative immunohistochemical image of Ki67 staining. Scale bar: 0.5 mm/0.1 mm.

I. Quantification of proliferative index (Ki67 staining) in rats injected with DAOY cells transfected with either control shRNA or shRNA against Fbxl17 (mean ± SEM; n = 5; unpaired t-test, *p<0.05).
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 7

Figure 7. Fbxl17-Sufu axis is altered in medulloblastoma

A-C. mRNA expression of Sufu, Gli1 and Fbxl17 detected in 285 medulloblastoma samples (GEO accession: GSE37382), stratified by tumor subtype. Level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8. Analysis using one-way anova gave comparable results.

D. Expression of Gli1 and Fbxl17 in the 285 patients is shown colored by subgroup; level of significance (p) for Kruskal-Wallis (KW) rank test is p<10-8, and Spearman’s rank correlation (R=0.5640) is indicated.

E. Detection of HA-tagged Sufu wild type (WT) and HA-tagged Sufu S352F protein levels in NIH3T3 cells after cycloheximide (CHX) treatment. Where indicated, cells were transfected with a non- targeting siRNA (Control) or two siRNA to Fbxl17 (1) and (2).

F. Assessment of Hh pathway activation using Gli1-luciferase reporter assay in MEFs Sufu-/- upon reintroduction of HA-tagged Sufu WT or Sufu mutant S352F using a retroviral system. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).
Functional interplay between c-Myc and Max in B lymphocyte differentiation (2018)
Figure 1

Figure 1. c-Myc/Max in B lymphocyte differentiation.

A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.

B, C, D Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM (B, C) or spleen (B, D) of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-), spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.

E Schematic representation of the gated populations in B and D flow cytometry plots.

F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry as in B, C, D. Experiment representative of at least three independent experiments.

I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.

J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.
Functional interplay between c-Myc and Max in B lymphocyte differentiation (2018)
Figure 2

Figure 2. Role of c-Myc/Max in B lymphocyte function and the immune response.

A, B, C, D Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) and B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.

E Spleen B220+GFP+ B lymphocytes from (A) or (C) were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis).

F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E.

G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0.

H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments.

I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.

J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Functional interplay between c-Myc and Max in B lymphocyte differentiation (2018)
Figure 3

Figure 3. Distinct roles of c-Myc and Max in the proliferation in mature B lymphocytes.

A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments

B Cell counts from the cultures in A and C (n=5).

C Photographs of B cell cultures from A and B. Scale bar, 500 µm. 4× magnification.

D Forward scatter (FSC) histograms of activated B lymphocytes from A. An arbitrary dashed line is shown for comparison.

E Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated as in A and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKO

F, G Representation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated as in A for 48 h. Scale bar, 10 µm.

H Fork rate of DNA fibres from primary B lymphocytes sorted and activated as in G. Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01

I Western blotting of DNA replication proteins in activated B lymphocytes from F and G. Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.
Functional interplay between c-Myc and Max in B lymphocyte differentiation (2018)
Figure 4

Figure 4. RNA-sequencing analyses in mature B lymphocytes.

A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.

B Venn diagrams showing the number of common DEGs in all conditions.

C Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.

D Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown. Colour code is the same as C.
Functional interplay between c-Myc and Max in B lymphocyte differentiation (2018)
Figure 5

Figure 5. Major up- and down regulated pathways in mature B lymphocytes.

A Enrichment analysis of KEGG pathways (FDR <0.05) of up- or down-regulated genes common for all three conditions (+/-1.5-FC and p<0.01). Relevant pathways are labelled in bold.

B Heatmaps of genes involved in the relevant pathways indicated in A. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.

C qPCR analysis of up- and down-regulated genes from B. Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated as in Fig 4A. Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.

Expanded view figure legends
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 1

Figure 1: TGF-β regulates S100A9 expression and its subcellular localization in Langerhans cells (LC)

RNA-Seq analysis was performed using total RNA extracted from Human primary monocyte-derived LC (MoLC), DC (MoDC), and MoDC treated with TGF-β for 24h (MoDC-TGF-β).

(A) Heat map showing differential expression of significant TGF-β regulated genes in MoDC, MoLC and TGF-β-treated-MoDC of independent experiments from three donors (n=3). Fold change normalization to autologous MoDC was done on Log2-transformed normalized FPKM data and represented by Heat map. Row clusterization was applied based on (Log2) Fold Change values. Green and red shades correspond to increased or decreased gene expression levels respectively.

(B) RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors.

(C) S100A8 and S100A9 expression were determined by western blotting of lysates from MoLC, MoDC and MoDC treated with TGF-β for 24h or (D) from cell lines as indicated. Loading controls were done with GAPDH or actin antibodies. Human primary cells results are representative of three donors and the experiment shown is representative of three (n=3).

(E) Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 2

Figure 2: S100A9 expression inversely correlates with MoLC maturation and susceptibility to HIV-1 infection

(A) MoLC from two different donors (donor #26 and #30) were left untreated (NT) or incubated with 1µg/ml of LPS for indicated time points before infection with HIV-GFP vectors for 48h. The percentage of GFP+ cells was analysed by flow cytometry (left dot-plots) and a graph representing the fold change of GFP+ cells, normalized to untreated infected MoLC is shown on the right.

(B) Non-treated (NT) and LPS-treated MoLC, as before, were analysed by flow cytometry for the expression of CD83 and HLA-DR.

(C) RT-qPCR and (D) western blot analyses of S100A9 expression in MoLC from the two donors treated as above. RT-qPCR data were represented as means ± SD.

(E) MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 4

Figure 4: S100A9 expression in human epidermal LC (eLC) restricts HIV-derived LV transduction

(A) MoLC, dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis. Scale bar: 5µm.

(B) Representative experiment of human epidermal cells analysed by flow cytometry for the expression of langerin, CD1a and S100A9 (Skin samples from n=2 donors). The enriched CD1a+/langerin+ population representing human eLC is shown (black circle).

(C) Lysates from CD1a+ and CD1a- cells from human skin dermis or epidermis (Epi) were subjected to immunoblotting for the expression of S100A9 proteins. Loading control was done with anti-actin (n=2).

(D) RT-qPCR analysis of S100A9 expression in human CD1a+ epidermal cells transfected with siCTR or siS100A9 (skin samples from n=2 donors). RT-qPCR data were represented as means ± SD.

(E) Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.

When applicable, student t-test was performed to determine statistical significance (***P<0.001).
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 5

Figure 5: S100A9 expression inhibits wild-type HIV-1 infection of MoLC and eLC

(A) Epidermal cells transfected with control siRNA (siCtrl) or S100A9-targetting siRNA (siS100A9) were challenged for 72h with 200 ng P24 of HIV-1-R5 (left dot-plots) or HIV-1 primary isolate (right dot-plots). Cells were fixed, permeabilised and stained with langerin (Y-axis) and HIV Gag P24 (X-axis) fluorescently coupled antibodies. Samples were acquired on a Flow cytometer. The experiment shown is representative of four donors for HIV-1-R5 (n=4) and five donors for HIV-1 primary isolates (n=5). (B) The same as above was done but on MoLC gated by size (FSC). The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with (C) HIV-1-R5 and (D) HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 6

Figure 6: Ectopic expression of human S100A9 in HEK293T cells inhibits HIV infection

(A, B) S100A9 expression was analysed by (A) immunoblotting or (B) flow cytometry in stably transduced HEK293T cells using anti-myc and anti-S100A9 as indicated. Immunoblot loading controls were done with anti-actin. Flow cytometry negative and positive controls were done with non-transduced parental cells (in grey) and HEK293T cells stably expressing the red emitting fluorescent protein E2C (E2C-Far Red), respectively. Experiments shown are representative of three (n=3).

(C) HEK293T were infected with HIV-Luc for 48h (10ng of p24). Graphs represent fold RLU means ± SD of infected S100A9-expressing cells normalized to infected E2C-expressing cells from duplicates of three independent experiments (n=3).

(D) E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.

:When applicable, student t-test was performed to determine statistical significance (*P<0.1***P<0.001).
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 7

Figure 7: Ectopic human S100A9 expression in CD4+ T lymphoid cell lines potently restricts X4-tropic wild-type HIV-1 infection

)(A) E2C- and S100A9-expressing CEM and (D) E2C- and S100A9-expressing Jurkat CD4+ T cells were analysed for the expression of E2C and S100A9 by flow cytometry . (B, E) Cells from above were infected with X4-tropic HIV-1 for 72h and processed for flow cytometry analysis upon staining with anti-HIV Gag p24-FITC. Dot-plots from representative experiments of (B) CEM or (E) Jurkat infected with X4-tropic HIV-1 (50ng of p24 per 105 cells) are shown. Control cells (NI) correspond to non-infected E2C-expressing cells. (C, F) The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 8

Figure 8: Human S100A9 expression restricts ex vivo MMLV replication and inhibits MMLV RTase activity in vitro

(A) MDTF cells were transfected with an empty vector (pCDNA3.1) or a vector expressing myc-tagged human S100A9 whose expression was analysed by Western-blotting with anti-myc 48h post-transfection. (B) MDTF cells were then challenged with MMLV-YFP vectors (MOI 1) for 48h. Flow cytometry dot-plots from a representative experiment are shown.

(C) Human S100A9 expression in CHO cells was analysed by immunoblotting or flow cytometry as in Figure 7.

(D) E2C- and S100A9-expressing CHO cells were challenged with indicated MOI of MMLV-YFP vectors for 24h or 48h. The percentages of YFP+ CHO infected cells analysed by flow cytometry and data were represented on graphs.
Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells (2021)
Figure 9

Figure 9: Human S100A9 negatively regulates HIV-1 reverse transcription

(A) MoLC were transfected with siCTR or siS100A9 and infected with HIV-GFP vectors for 24h. DNA was extracted and copies of late reverse transcripts were quantified by qPCR. Shown data were collected from experimental triplicates ± SD of four different donors (n=4).

(B) MoLC or eLC previously transfected with control siRNA (siCtrl) or S100A9-targetting siRNA (siS100A9) were challenged with 200 ng P24 of HIV-1-R5 for 24h. Nevirapine (10μM) was used as a control of RT inhibition. Total DNA was then extracted and late RT products were quantified by qPCR and normalized as copies/100ng DNA using a standard curve based on the serial dilution of a defined and linearized HIV-1 plasmid. Duplicates from three different donors are shown (n=3). Nevirapine (10μM) treatment was performed once on each donor to control RT inhibition. Data were represented as means ± SD.

(C) The same experimental procedure as above was done with HIV-1 primary isolates. Data in triplicates ± SD from three different donors (n=3) were collected and represented as bar charts.

(D) E2C-expressing (upper dot-plot) and S100A9-expressing (lower dot-plot) THP-1 cells were controlled for E2C and S100A9 expression by flow cytometry upon analysis of fluorescence as indicated in the X axis label. Parental untransduced THP-1 cells were used as control cells (gray dots). The experiment shown is representative of 3.

(E) E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown. (F) Means ± SD of duplicates from two independent experiments are presented in the graph (n=2).

When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).