EMBO/soda-nel huggingface dataset:
Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders (2010)
Figure 4

The LSD endolysosomal membrane contains increased amount of cholesterol‐enriched regions. (A) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots. (B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT. (C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions. (D) Equal aliquots from either DRMs or soluble fractions were pooled, the protein content determined and displayed as percentage of total protein in DRM and soluble gradient regions. (E) Immunoblotting profiles of the transferrin receptor and LAMP1 in the sucrose gradient. Values represent the mean±s.e.m. values of three experiments (A–D). *P0.05, Student's t‐test: (A–C): WT versus MSD and WT versus MPS‐IIIA; (D): WT versus MSD and WT versus MPS‐IIIA for each fraction. Scale bar: 0.3 μm (B).
Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders (2010)
Figure 5

SNAREs are sequestered by cholesterol within endolysosomal membranes. (A) VAMP7, Vti1b and syntaxin 7 distributions in endo‐lysosomal membranes from MSD, MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions. To simplify the analysis the DRM, the intermediate‐I, the intermediate‐II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting. SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment. In MSD and MPS‐IIIA MEFs, all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes. Cholesterol modulation results in a change of SNARE distribution. (B) The percentage of each analysed SNARE observed in DRM fractions was quantified from blots (ImageJ densitometry analysis) and displayed as relative amount versus WT. (C, E) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in (C) endolysosomal membranes and (E) total cell lysates from WT (untreated and cholesterol treated) and MSD MEFs (untreated and MβCD treated). In the graphs, the protein levels were displayed as relative amount versus WT. (D) WT and MSD MEFs transfected with GFP–VAMP7 were stained with anti‐GFP for immuno‐EM. The enlarged image shows internalization of GFP–VAMP7 particles (arrow). (F) Syntaxin 5, Sec22 and SNAP23 distribution in total membrane derived from control WT and MSD MEFs. Syntaxin 5 immunoblot shows two bands (*, 35 kDa and **, 42 kDa) corresponding to the two isoforms of the protein. (G) Distribution profile of Rab7 in WT and MSD lysosomal membranes. Values represent the mean±s.e.m. values of three independent experiments (B, C, E). *P0.05, Student's t‐test: (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD, and MPS‐IIIA versus MβCD for each analysed SNARE; (C, E): WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each analysed protein. Scale bar: 0.3 μm (D).
Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders (2010)
Figure 6

SNAREs are locked in an assembled form in LSD endolysosomal membranes. (A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50–60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart). (B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown. (C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs. (D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.
Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders (2010)
Figure 7

Cholesterol levels affect SNARE trafficking. (A) MSD, MPS‐IIIA and WT MEFs were subjected to a triple labelling with anti‐VAMP7, anti‐Vti1b and anti‐LAMP1 antibodies. The merges between VAMP7 and Vti1b (double merges in yellow) and between VAMP7, Vti1b and LAMP1 (triple merges in white) are shown (see also enlarged images showing the extent of co‐localization in different regions of the cells). The VAMP7–Vti1b co‐localization was quantified in 15 different cells and displayed as % of Vti1b co‐localizing with VAMP7 (means±s.e.m.). (B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells. (C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP–Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells. A summary of t1/2 values is also shown. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B): WT versus MSD, WT versus MPS‐IIIA, WT versus WT+cholesterol, MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD); (C): WT versus MSD, WT versus WT+cholesterol, MSD versus MSD+MβCD. Scale bar: 10 μm (A, B).
Quorum sensing governs collective dendritic cell activation in vivo (2021)
Figure 2

Figure 2. Cellular density influences dendritic cell activation by poly(I:C) in vitro

(A‑B) BMDCs were plated at different densities and stimulated with poly(I:C) for 24h or left unstimulated. DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry. (A) Representative dot plots showing CD86 and CD40 expression. (B) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity (gMFI) over unstimulated controls. Data are pooled from 3 independent experiments.

(C) MutuDCs were plated at different cell densities and stimulated with poly(I:C) or left unstimulated. The graph shows the upregulation of CD86 as a fold change in gMFI over unstimulated cells. Data are pooled from 4 independent experiments.

(D) Flow cytometric quantification of CD86 and CD40 upregulation on WT (blue) or Ifnar‑/‑ (green) BMDCs expressed as a fold change in gMFI over unstimulated controls.

(E) CD86 and CD40 expression was quantified on BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) in the presence (red dots) or absence (blue dots) of anti‑IFNAR1 blocking antibody. Data are pooled from 2 independent experiments.

(F) CD86 and CD40 expression was quantified on BMDCs plated at low density (2x103 cells/cm2) and stimulated with poly(I:C) (dark blue dots), IFN‑α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments.

(G) IFN‑α (left) and IFN‑β (right) production was quantified by ELISA for BMDCs plated at different densities and stimulated or not with poly(I:C). Dotted lines represent the ELISA threshold of detection. Data are pooled from 2 independent experiments.

Data information: Black lines indicate mean values. Dotted lines in B, C, D, E and F represent a fold change equal to 1. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns; non-significant, one-way ANOVA (B, C, and F) and unpaired t-test (D, E and G)).
Quorum sensing governs collective dendritic cell activation in vivo (2021)
Figure 3

Figure 3. The density of type I IFN‑producing cells regulates DC activation by poly(I:C) in vitro

(A) IFN‑α (left) and IFN‑β (right) production by WT and Irf3-/-Irf7-/- BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) (blue dots) or left unstimulated (grey dots). Dotted lines represent the ELISA threshold of detection. Data are representative of 2 independent experiments.

(B) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) or left unstimulated. Data are pooled from 2 independent experiments.

(C) CD86 and CD40 expression on Irf3-/-Irf7-/- BMDCs plated at 2x105 cells/cm2 and stimulated with poly(I:C) (dark blue dots), IFN-α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments.

(D-E) WT and Irf3‑/‑Irf7‑/‑ BMDCs were mixed at different ratios for a total number of 2x105 cells/cm2 and stimulated with poly(I:C) or left unstimulated. (D) Representative dot plots showing CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) BMDCs. (E) Flow cytometric quantification of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) BMDCs expressed as a fold change in gMFI over unstimulated controls.

(F) CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) BMDCs expressed as a fold change in the proportion of CD86+ CD40+ cells over unstimulated controls. Data are pooled from 2 independent experiments.

Data information: Black lines indicate mean values. Dotted lines in B, C, E and F represent a fold change equal to 1. (**p<0.01, ***p<0.001, ****p<0.0001, ns; non‑significant, unpaired t‑test (A, B, E and F) and one‑way ANOVA (C)).
Quorum sensing governs collective dendritic cell activation in vivo (2021)
Figure 4

Figure 4. A quorum‑based mechanism governs DC activation in vivo
(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments.

(B) Experimental set-up. Irf3‑/‑Irf7‑/‑ recipient mice were lethally irradiated and reconstituted with WT (GFP+) and Irf3‑/‑Irf7‑/‑ (GFP‑) bone marrow cells (BMCs) mixed at different ratios. Chimeras were injected in the footpad 4 weeks later with poly(I:C) and WT and Irf3‑/‑Irf7‑/‑ DCs from non‑draining and draining popliteal lymph nodes were analyzed 24h after poly(I:C) injection by flow cytometry.

(C) Microscopy images of sliced draining lymph nodes in CD11c‑eYFP:Irf3‑/‑Irf7‑/‑ chimeras with 90:10 (left) and 5:95 (right) ratios. Type 1 IFN‑competent cells appear in green. Mosaic images were generated by acquisition of overlapping imaging fields. Scale bars represent 25 µm. (D-E) CD86 and CD40 expression on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) DCs from draining lymph nodes of mixed‑bone marrow chimeras with indicated WT:Irf3‑/‑Irf7‑/‑ ratios.

(D) Representative dot plots of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs of chimeras injected with PBS or poly(I:C).

(E) Flow cytometric quantification of CD86 and CD40 expression on WT and Irf3‑/‑Irf7‑/‑ DCs in chimeras injected with PBS or poly(I:C). Results from 5:95 and 10:90 chimeras were similar and pooled. Data are pooled from 3 independent experiments.

Data information: Each dot represents one mouse. Black lines indicate mean values. Dotted lines in A and E represent a fold change equal to 1. (**p<0.01, ***p<0.001, ****p<0.0001, ns; non‑significant, unpaired t-test).
Quorum sensing governs collective dendritic cell activation in vivo (2021)
Figure 5

Figure 5. Optimal DC activation requires prolonged exposure to type I IFN

(A) Experimental set‑up. BMDCs cultured at low density (2x103 cells/cm2) were stimulated with poly(I:C) and IFN‑α was added at various time points. IFNAR signaling was interrupted using anti‑IFNAR1 blocking antibody at the indicated time point. BMDC activation was measured 24h after poly(I:C) stimulation by flow cytometry.

(B) Representative dot plots showing CD86 and CD40 expression by low‑density BMDCs stimulated with poly(I:C) for different periods of IFN‑α exposure.

(C) Quantification of CD86 and CD40 upregulation by BMDCs expressed as a fold change in gMFI over unstimulated controls. Data are pooled from 2 independent experiments. Black lines indicate mean values. Dotted lines represent a fold change equal to 1. (**p<0.01, ***p<0.001, ****p<0.0001, one‑way ANOVA).
Quorum sensing governs collective dendritic cell activation in vivo (2021)
Figure 6

Figure 6. Collective DC activation regulates inflammation and T cell immunity

(A‑B) Monocyte recruitment in the draining lymph node requires collective DC activation (A) Representative dot plots showing the percentage of monocytes (CD11b+ Ly6Chigh cells, black box) present in popliteal dLN of WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) (middle and right) or with PBS (left). CD11b+ Ly6Clow/int cells corresponded to neutrophils and were not included in the analysis. (B) Flow cytometric quantification of monocyte numbers in the lymph nodes of mice injected with poly(I:C) expressed as a fold change over PBS controls. Data are pooled from 2 independent experiments.

(C‑D) Activation of NK cells in the draining lymph node requires collective DC activation. (C) Representative histograms showing granzyme B and CD69 expression by NK 1.1+ Ly6Clow cells in dLN of 90:10 (dark blue) and 5:95 (grey) WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) or with PBS (black line). (D) Quantification of granzyme B intracellular content (left) and CD69 expression (right) in NK1.1+ Ly6Clow cells of chimeric mice injected with poly(I:C), expressed as a fold change in gMFI over PBS‑injected controls. Data are pooled from 2 independent experiments.

(E‑F) Crosspriming of CD8+ T cells requires DC activation and type I IFN. (E) Representative dot plots showing the percentage of CD44high Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of WT and Irf3‑/‑Irf7‑/‑ mice injected with PBS, OVA or a mixture of OVA plus poly(I:C). (F) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of WT mice injected with PBS (grey dots), OVA (black dots), OVA plus poly(I:C) (blue dots) and in draining lymph node of Irf3‑/‑Irf7‑/‑ mice injected with OVA plus poly(I:C) (orange dots). Data are pooled from 2 independent experiments.

(G‑H) Collective DC activation is essential for the generation of T cell responses. (G) Representative dot plots showing the percentage of CD44high tetramer H‑2Kb‑OVA+ CD8+ T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA+poly(I:C). (H) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of 90:10 and 5:95 (WT:Irf3‑/‑Irf7‑/‑) chimeric mice injected with PBS or OVA+poly(I:C). Data are pooled from 2 independent experiments.

Data information: Each dot represents one mouse. Black lines indicate mean values. Dotted lines indicate fold change equal to 1. (**p<0.01, ***p<0.001, ns; non‑significant, unpaired t‑test (B and D) and one‑way ANOVA (F and H).
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 2

Figure 2: Exposure to CDK7i induces melanoma dedifferentiation

A. Volcano plots were used to demonstrate differentially expressed genes as determined by RNA-seq in either MM047CDK7i-R vs MM047 (top), MM074CDK7i-R vs MM074 (middle) or MM074BRAFi-R vs MM074 (bottom). Red dots show significantly over-(top) or under-(bottom) represented mRNA in drug-resistant cells compared to parental cells. All data were evaluated with the DESeq2 R package. The value for a given gene is the normalized gene expression value relative to the mean of all samples belonging to the same condition.

B. Proportional Venn diagrams indicating the number of up- (left) and down- (right) regulated genes in MM047CDK7i-R and MM074CDK7i-R compared to the parental MM047 and MM074, respectively. The number of genes overlapping between the data sets is indicated. 261 genes were found up-regulated and 241 down-regulated in MM047CDK7i-R and MM074CDK7i-R. Hypergeometric p-value is indicated.

C. Genes characterizing the melanocytic-type and mesenchymal-like transcription signatures (Widmer et al., 2012) have been plotted on a Heatmap and are shown in relation to their expression in different melanoma cells. RPKM values are represented as z-score. The group of genes related to pigmentation has been highlighted in red. The color key shows the log2 expression values. Yellow color stands for high expression and dark violet for low expression.

D. Protein lysates from MM074, MM074BRAFi-R or MM074CDK7i-R were immuno-blotted for indicated proteins. Molecular sizes of the proteins are indicated (kDa). The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control.
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 3

Figure 3: GATA6 and its regulon are expressed in CDK7i-tolerant melanoma cells

A. qRT-PCR analysis showing average TBP-normalized expression of GATA6 in the indicated cells.

B. Protein lysates from the indicated cells were immuno-blotted for the indicated proteins. Molecular sizes of the proteins are indicated in kDa. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control.

C. UMAP (Uniform Manifold Approximation and Projection) dimension reduction representative of GATA6 regulon expression in MM011, MM029, MM047, MM074 and MM099 from Wouters dataset (Wouters et al., 2020). UMAP is colored according to the continuous GATA6 AUCell values (from 0 to 0.2).

D. qRT-PCR analysis showing average TBP-normalized expression of AMIGO2, SERPINE1 and ABCG2 in the indicated cells.

E-H. qRT-PCR analysis showing average TBP-normalized expression of GATA6 (E), AMIGO2 (F), SERPINE1 (G) and ABCG2 (H) in the indicated cells treated with either siCTL or siGATA6 for 72h.

I. MM011 and MM029 were treated with either siCTL or siGATA6 for 72h. Cell proliferation was analysed using CellTrace staining and flow cytometry in the indicated cell lines and the % of slow proliferating cells is shown for each condition.

J. MM029 (left) and MM099 (right) were pre-treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells.

Data information: In (A, D-J), data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 4

Figure 4: ABCG2 is involved in multidrug tolerance in melanoma cells

A. qRT-PCR analysis showing average TBP-normalized expression of ABCB1, ABCC3 and ABCG2 in the indicated cells.

B. Protein lysates from the indicated cells were immuno-blotted for the indicated proteins. Molecular masses of the proteins are indicated in kDa. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software, and normalized to each relative βTUBULIN control.

C-D. MM099 (c) and MM029 (d) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells.

E-F. MM099 (e) and MM029 (f) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells.

Data information: In (A), data are presented as mean values + SD for six replicates (n=6). In (C-F), data are presented as mean values + SD for three replicates (n=3). IC50 for each cell line is indicated.
CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma (2021)
Figure 5

Figure 5: CDK7i induced inhibition of MITF and SOX10 and release of GATA6 expression

A-B. Gene track of CDK7 occupancy at MITF (a) or SOX10 (b) loci in 501melBIO-FLAG:CDK7 cell line. Gene tracks of H2A.Z, BRG1, MITF, SOX10 and H3K27ac (GSE94488 and GSE61967) at the same loci in parental 501mel are indicated. SE is denoted by a red opened square. H3K27ac deposition is also shown in Hair Follicle Melanocytes (HFM) (GSE94488).

C-E. qRT-PCR analysis showing average TBP-normalized fold expression of MITF (C), SOX10 (D) and GATA6 (E) in 501mel treated either with DMSO/THZ1 (50nM) (upper) or DMSO/JQ1 (10μM) (lower) for 24h.

Data information: In (C-E), data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.