EMBO/soda-nel huggingface dataset:
XIAP promotes melanoma growth by inducing tumour neutrophil infiltration (2022)
Figure 1

Fig 1: XIAP promotes tumour growth without interfering with tumour cell apoptosis

A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines. Actin was used as a loading control.

B Subcutaneous melanoma tumour growth in BL/6 WT mice (n=5) using B16F1 WT and XIAPKO (clone1 and 2). Data are mean ± s.e.m. Two-way analysis of variance (ANOVA) followed by Dunnett's post-analysis.

C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5). Data are presented as mean ± s.e.m. Mann-Whitney, two-tailed test analysis.

D qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in B16F1 WT cells plotted vs B16F1-XIAPKO cells (clone1 and 2). Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.

E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr (control) or siXIAP and Western blot analysis of the corresponding cell lysates (bottom). One-way analysis of variance (ANOVA) followed by Dunnett's post-analysis.

F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells. Unpaired t test, two tailed analysis.

G qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in HEK293T cells overexpressing myc-XIAP plotted vs HEK293T cells overexpressing myc-XIAPΔRING. Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.

H Human cytokines array analysis of HEK293T cells transfected with myc-XIAP or myc-XIAPΔRING. Medium was changed after 16 h and the supernatant was collected after 48 h. Pre-spotted nitrocellulose membranes were incubated with the supernatants overnight at 4°C.

Data information: In E and F, data are presented as mean ± SD. Dots represent individual biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant.
XIAP promotes melanoma growth by inducing tumour neutrophil infiltration (2022)
Figure 2

Fig 2: XIAP mediates IL8 secretion in melanoma by involving TAB1 and RIPK2

A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments.

B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue). Scale bar 20 µm.

C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.

D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.

E IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA.

F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.

G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.

Data information: In E, F and G, dots represent individual biological replicates. Data are presented as mean ± SD. Unpaired t test, two-tailed test analysis (F) or One-way analysis of variance (ANOVA) followed by Dunnett's post-analysis (E and G). *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001.
XIAP promotes melanoma growth by inducing tumour neutrophil infiltration (2022)
Figure 4

Fig 4: XIAP-RIPK2-TAB1 axis supports neutrophil infiltration and melanoma growth in B16 mouse melanoma

A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored. The numbers of melanomas with different intensities were plotted into the table as a percentage of the total. Specific staining intensity for XIAP and CD177, corresponding to relative amounts of infiltrated neutrophils, was arbitrarily set as the following: -, not expressed; +, low; ++, moderate; and +++, strong expression. Representative IHC pictures (right panel). Scale bar 100 µm.

B Representative IF (neutrophils staining) in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO tumours (Scale bar 50 µm) and quantification of Ly6G+ cells. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).

C B16F1 WT, XIAPKO (clone 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5). Mice were intra-peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells. Data are mean ± s.e.m. Two-way analysis of variance (ANOVA) followed by Dunnett's post-analysis.

D Representative IF (neutrophils) scale bar 50 µm and quantification of Ly6G+ cells in tumours from C. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (4 mice per genotype) was represented. Dots represent individual mice (n=4).

Data information: In B and D, data are presented as mean ± s.e.m. One-way analysis of variance (ANOVA) followed by Dunnett's post-analysis. **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant.
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis (2014)
Figure 1

Experimental outline of the cell surface labeling and preparation for mass spectrometry

Immunostaining of HeLa cells probed for FITC‐labeled streptavidin and DNA and the merge streptavidin (red) and DNA (blue). Cell surface was labeled using amine biotinylation. Scale bar, 10 μm.
Western blot analysis of cell surface preparation. The surface‐biotinylated cells were lysed using a detergent containing lysis buffer, incubated with and then eluted from streptavidin beads (i.e., cell sur. fraction). Cell surface fraction and the whole‐cell lysate were blotted against a cell surface protein, EGFR, and a cytoplasmic protein, actin.
Workflow of the cell surface proteome. Cell surface labeling and isolation of cell surface proteins in SILAC‐labeled interphase versus mitotic HeLa cells are shown.
Schematic of all identified proteins (blue circle) and cell surface proteins (pink circle). Cell surface/plasma membrane proteins were identified based their GO annotations; their distribution is shown in a pie chart (top).

Source data are available online for this figure.
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis (2014)
Figure 2

SILAC‐based quantitative analysis of cell surface proteins in interphase and mitosis

Histogram of the isotope ratio distribution before (left) and after (right) normalization and centering. The x‐axis shows the mitosis/interphase ratio on a log2 scale. Note that more cell surface proteins were recovered from the interphase cells prior to normalization.
Western blot confirmation of the mitosis‐selective cell surface localization of PCDH7. Western blotting analysis of cell surface protein preparation from mitosis and interphase cells. The whole‐cell lysate and elution from streptavidin beads were blotted against TFRC, EGFR, actin, and phospho‐Histone H3. TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation, while PCDH7 protein was enriched in mitotic cell surface preparation.
Western blot confirmation of the mitosis‐selective cell surface localization of PCDH1. Whole‐cell lysate samples were subjected to SDS–PAGE and blotted with the anti‐PCDH1, anti‐β‐actin, and anti‐phospho‐Histone H3 antibodies, as indicated on the left. Cell surface fraction samples were blotted with the anti‐PCDH1 and anti‐ EGFR antibodies.

Source data are available online for this figure.
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis (2014)
Figure 3

Mitosis‐selective cell surface localization of PCDH7

Tubulin and PCDH7 immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue. PCDH7 localizes to the mitotic cell surface (arrows). PCDH7 staining is barely detectable in the surrounding interphase cells. Scale bar, 5 μm.
Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis (n = 6, red) and interphase (n = 7, blue). Bars show mean ± SEM.
PCDH7‐EGFP‐transfected unsynchronized U2OS cells, stably expressing mCherry‐tubulin, at mitosis (top) and interphase (bottom). PCDH7, green; tubulin, red; DNA, blue. Scale bar, 10 μm.
Immunostaining of HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes (green) probed for tubulin (red) and DNA (blue). Top panel shows mitotic cell those were treated with 5 μM STC for 10 h, and bottom panel shows interphase cells. Scale bar, 10 μm.
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis (2014)
Figure 4

Cytoplasmic domain of PCDH7 is required for its cell cycle‐dependent cell surface localization

Confocal images representing the localization of PCDH7. U2OS cells expressing full‐length (WT) PCDH7 at mitosis (top) and interphase (middle). Cytoplasmic deleted (CytΔ) PCDH7‐expressing cells at mitosis (middle) and interphase (bottom). PCDH7, green; DNA, blue. Scale bar, 20 μm.
Quantification of PCDH7::GFP (WT and CytΔ) protein localization on the cell surface in mitosis [WT (n = 11), CytΔ (n = 6), red] and interphase [WT (n = 25), CytΔ (n = 20), blue]. Interphase cytΔ PCDH7 localizes to the cell surface more than WT (full length) PCDH7 (insets). Bars show mean ± SEM.
In interphase co‐localization between PCDH7 and ER marker KDEL. HeLa cells were transiently transfected with PCDH7::GFP (green) (ectopic), fixed and stained for ER marker KDEL (red) (interphase, top panel). Interphase (middle panel) and mitotic (bottom panel) HeLa cells were fixed and stained against (endogenous) PCDH7 and KDEL. Scale bar, 5 μm.
PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction. Whole‐cell lysate, cytoplasmic, endoplasmic, and mitochondrial fractions were blotted against an endoplasmic reticulum protein, KDEL, a cytoplasmic protein, actin, a mitochondrial protein Cox‐2 and PCDH7, PCDH1.

Source data are available online for this figure.
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis (2014)
Figure 5

PCDH7 is required on the cell cortex for building rounding pressure

PCDH7 antibody detects a single band of ˜110 kDa in luciferase RNAi (luc), PCDH1 RNAi HeLa cell lysates, but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells. α‐tubulin was used as a loading control (left). HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with luciferase RNAi (control) and 150 nM (1×) and 300 nM (2×) PCDH1 esiRNA and blotted against anti‐GFP, anti‐PCDH1, and anti‐actin antibodies. PCDH1 bands were reduced in PCDH1 RNAi cells, whereas mouse GFP::PCDH1 was not affected by the esiRNA treatment (right).
Rounding pressure plots of MYH9 (positive control, n = 7), luc (negative control, n = 12), PCDH7 (n = 11), PCDH1 (n = 12), and PCDH1 and PCDH7 (n = 11) RNAi cells. Bars show mean ± SEM (left). Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with MYH9 (positive control, n = 7), luc (negative control, n = 14), PCDH1 (n = 13) esiRNA. *P 0.05; **P 0.01; ***P 0.001; ns, P > 0.05 (non‐significant).

Source data are available online for this figure.
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis (2014)
Figure 6

Protocadherin‐7 as a mitotic marker

Workflow of flow cytometry analysis.
Flow cytometry analysis of HeLa cells treated with anti‐PCDH7 antibody, fixed and stained for phospho‐Histone H3 and DNA. Forty‐two percent of unsorted HeLa cella are in G2/M phase. When cells are sorted based on their phospho‐Histone H3 (middle) expression, phospho‐Histone H3+ (high expression) cells are preferentially mitotic (87.3% in G2/M phase) and 42% of those are PCDH7+. Similarly, PCDH7+ (high expression) cells are preferentially mitotic (82.1% in G2/M phase) and 79% of those are phospho‐Histone H3+ (bottom).
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 1

Figure 1: Clinical and molecular studies on PITRM1R183Q mutant patients.

A: family tree. Affected subjects are represented by filled shapes. The index case is II-2 (arrow).

B: brain MRI images of II-1 (FLAIR). A: sagittal interemispheric sequence showing profound cerebellar atrophy (arrow); B: transverse sequence showing involvement of the dentate nuclei of cerebellum; C: coronal sequence revealing cortical atrophy, a wide third ventricle, and high signal changes in the thalami, particularly on the left (arrow), D: the thalamic lesion is indicated by an arrow.

C: histochemical staining of skeletal muscle from II-2. Combined COX/SDH staining shows scattered COX negative (blue) fibres (arrows). The black bar corresponds to 100 µm.

D: respiratory chain complex activities in skeletal muscle. Specific activities (nmol min-1 mg-1) of complex I, II and IV are reduced in II-2 (red) compared to the controls mean (blue); CS activity is also low (<50% of controls) suggesting a reduction of mitochondrial mass (see main text for further details). Each activity was measured in triplicate.

E: Western blot analysis of PITRM1 in primary fibroblasts (left) and skeletal muscle (right) of controls (CTR) and subject II-2. Densitometric quantification using the Genetools software is shown below the blots. In blue is PITRM1WT and in red PITRM1R183Q.

F: proteolytic activity on two fluorescent oligopeptides (AnaSpec), reported at the bottom of each histogram, by 6-His tagged PITRM1WT and PITRM1R183Q proteins expressed in E. coli and affinity-purified by Ni-agarose chromatography. Values are expressed as arbitrary units per sec per µg of protein (a.u. s-1µg-1). Experiments were performed in duplicate.

G: degradation rate of Aβ1-42 by purified 6-His tagged PITRM1WT and PITRM1R183Q proteins. Quantification of these experiments is displayed below the blots. Experiments were performed in duplicate.
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 2

Figure 2. Characterization of immortalized cells.

A: cell growth in glucose (GLU) and galactose (GAL). C29V, C47V, TpLV: immortalized fibroblast cell lines from control individuals; PITRM1V: immortalized fibroblasts from subject II-2, carrying the PITRM1R183Q mutation. pLKO.1: empty vector; sh38: shRNA38; sh41: shRNA41. Each cell line was measured six times. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA post-hoc Bonferroni test ***p<0.001.

B: Western-blot analysis of PITRM1 in C29V cells transduced with the empty vector plKO.1 and the two shRNA, sh38 and sh41. SDHB and β-Actin are used as loading controls.

C: co-localization of PITRM1 (red) with TOM20 (T20, green) in human fibroblast cells from a control (CTR) and subject II-2. Note that the intensity of PITRM1 immunofluorescence is much lower in II-2 cells than in CTR cells (see main text for further details). Nuclei are stained in blue by DAPI. A white bar corresponds to 10 µM.

D: mitochondrial membrane potential (P). In healthy cells with high mitochondrial P, JC-1 forms complexes known as J-aggregates that show an intense red fluorescence. On the contrary in unhealthy cells with low P, JC-1 remains in the monomeric form, showing only green fluorescence. Values are referred to as % of those of the control means, taken as 100%. The results are the mean of 3 independent experiments. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA **p <0.01; ****p<0.0001.

Exact p values are reported in Table EV1.
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 3

Figure 3. Modelling the cym1R163Q mutation in Saccharomyces cerevisiae.

A: oxidative growth. W303-1Bcym1Δ strains harbouring the wild-type CYM1 allele (CYM1wt), the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells/ml. Five microliters of each dilution were spotted on SC agar plates without uracil, supplemented with 2% glucose, 2% glycerol or 2% ethanol. Plates were incubated at 37°C for 3-7 days.

B: oxygen consumption rate (OCR). Cells grown at 37°C SC medium without uracil were supplemented with 0.5% glucose. Values were normalized to the OCR of the CYM1wt strain (49 nmol O2 min−1 mg−1) and represented as the mean of at least three values ± SD. Statistical analysis was by unpaired, two-tail Student’s t test: ***p<0.001.

C: reduced versus oxidized cytochrome spectra. Peaks at 550, 560 and 602 nm correspond to cytochromes c, b and aa3, respectively. The height of each peak relative to the baseline is an index of cytochrome content.

D: respiratory chain complex activities. Biochemical activities of Succinate Quinone DCPIP Reductase, SQDR (CII), NADH-cytochrome c oxidoreductase activity NCCR (CIII), and cytochrome c oxidase (CIV) were measured on a mitochondrial-enriched fraction from cells grown at 37°C as in panel B. Values were normalized to that of CYM1wt strain and represented as the mean of three independent experiments ± SD. Statistical analysis was by unpaired, two-tail Student’s t test: **p<0.01; ***p<0.001.

E: Western-blot using an anti-HA monoclonal antibody visualizing the CYM1wt and cym1R163Q recombinant proteins both fused in frame with the HA epitope on the C-terminus. Western-blot analysis on total protein extract. Total protein extracts were obtained by strains expressing HA-tagged CYM1wt and cym1R163Q. PGK was used as a loading control and signals were normalized to the wt. The quantification was performed on 5 independent blots.

F: prolonged exposure of a Western-blot containing the CYM1wt and cym1R163Q recombinant proteins reveals the presence of a band corresponding to a degradation product in the cym1R163Q lanes (arrow).

G: Western blot of Aβ1-42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37°C in SC medium supplemented with 0.15% glucose and 2% galactose. VDAC is used as a loading control. Each experiment was performed in triplicate.

Exact p values are reported in Table EV1.
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 4

Figure 4. Behavioural studies of Pitrm1+/- mouse.

A: Western-blot analysis of Pitrm1 protein in brain, muscle and liver of two 4-mo male Pitrm1+/- mice and two Pitrm1+/+ littermates. Densitometric analysis is reported in the histograms below the blots. Pitrm1+/+ is in blue, Pitrm1+/- in red. Error bars indicate ±SD

B: representative hindlimb-clasping phenomenon is shown in a 4 mo Pitrm1+/- male mouse, consisting in strong adduction of the hindlimbs when the animal is suspended by the tail; a littermate Pitrm1+/+ control displays the normal reflex, consisting in wide abduction of the limbs. All examined Pitrm1+/- animals displayed this abnormal reflex from 2 months of age.

C: rotarod test. Blue and red lines refer to Pitrm1+/+ (n=9) and Pitrm1+/- (n=7) 4 mo animals, respectively. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student’s t test. *p<0.05; ** p<0.01.

D: negative geotaxis test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student’s t test. * p<0.05.

E: cylinder test. The experiment was carried out on the same group of animals as in panel b. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student’s t test. *** p<0.001.

F: pole test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student’s t test. ***p<0.001.

Exact p values are reported in Table EV1.
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 5

Figure 5. Molecular and morphological analysis of Pitrm1+/- mouse brain.

A: Western-blot analysis of amyloid-precursor protein (APP) in 6 mo male mice. GAPDH was used as a loading control. Densitometric analysis on a total of 5 independent samples is shown in the histogram on the right.

B: morphological analysis of an AD subject and of Pitrm1+/- and Pitrm1+/+ mouse brains. TF: Thioflavin T (thalamus); CR: Congo-red (brain cortex); PL: polarised light (same sections as those stained with CR); Aβ1-42 immunostaining (pons); Ub: ubiquitin immunohistochemistry shown as brownish staining (brain cortex). The white bar indicates 20 µm.
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 6

Figure 6. Aβ1-42 mitochondrial import and degradation

A: upper panel: import of Aβ1-42 into mitochondria; lower panel: relative quantification from two independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the resulting value at 5 min without trypsin was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student’s t test. *p<0.05.

B: upper panel: pulse and chase experiment to assess clearance of Aβ1-42; lower panel: quantification from three independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the pulse value was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student’s t test. *p<0.05, **p<0.01.

Exact p values are reported in Table EV1.
Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration (2015)
Figure 7

Figure 7. Degradation of Aβ1-40 peptide in fibroblasts and MEFs.

A: MEFs from Pitrm1+/+, Pitrm1+/-, Mfn1-/-, Mfn2-/-, as well as h-PITRM1 R183Q and h- PITRM1 WT were exposed to Aβ1-40 peptide for 18h. Note that Aβ1-40 signal was still evident in Pitrm1+/- and h-PITRM1 R183Q cells.

B: quantification of three independent experiments similar to those shown in panel A. Statistical analysis was by unpaired, two-tail Student’s t test. **p<0.01, ***p<0.001.

Exact p values are reported in Table EV1.