EMBO/soda-nel huggingface dataset:
Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer (2019)
Figure 1

Figure 1. Expression of VPS4B is downregulated in CRC.

A Left panel, a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan-Cancer dataset. Vertical red line indicates the localization of VPS4B. Right panel, enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples. Deletions are marked in blue, amplified regions are marked in red. Both panels were generated with UCSC Xena browser (https://www.biorxiv.org/content/10.1101/326470v3).

B Analysis of VPS4B copy number alterations in TCGA Pan-Cancer dataset. Cancer types were sorted according to the mean VPS4B copy number after removing germline values.

C Scatter plot, analysis of VPS4B mRNA expression (number of transcripts per million) plotted against VPS4B copy number from TCGA CRC patient samples (n=376); plot generated using cBioPortal Gao et al, 2013(). Pie chart, summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma (TCGA, Provisional) dataset on cBioPortal (http://www.cbioportal.org/).

D qPCR analysis of VPS4B mRNA abundance in normal colon, adenoma and CRC samples. Adenocarcinoma (n=26); adenoma (n=42); normal colon (n=24). Green horizontal bars indicate means and red whiskers indicate SD. Differences were analyzed using the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test; ns - non-significant (p≥0.05), ****p<0.0001.

E Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in (F). 3+ - very intensive staining, 2+ - medium-intensive staining, 1+ - weak staining, 0 - no staining. Scale bar, 50 µm.

F Comparative analysis of VPS4B staining performed in 100 pairs of normal colon and matched CRC samples.

The exact p-values can be found in the source data for this figure.
Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer (2019)
Figure 3

Figure 3. Synthetic lethality between VPS4A and VPS4B inhibits growth of CRC cells in a mouse xenograft model.

A Schematic illustration of xenograft experiments with HCT116 VPS4B-/- cells having doxycycline (Dox)-inducible expression of shRNA targeting VPS4A.

B Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.

C Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.

D Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.

The exact p-values can be found in the source data for this figure.
Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer (2019)
Figure 5

Figure 5. Combined VPS4A+B depletion induces NF-κB signaling and caspase-dependent and -independent cell death pathways.

A Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.

B Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho-Rel and cleaved caspase 7), 9 (p52, caspase 8 and cleaved caspase 9), 7 (phosphorylated and total IκB) or 5 (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one-sample t-test (caspase 8, cleaved caspases 9, 7 and 3, total and phospho-IκBα) or Wilcoxon signed rank test (phospho-RelA and p52); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

C Immunoblot showing induction of the NF-κB pathway (p52) and apoptosis (cleaved caspase 7) in xenograft samples described in Fig 3C. cl - cleaved caspase. Vinculin was used as a loading control.

D Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.

The exact p-values can be found in the source data for this figure.
Synthetic lethality between VPS4A and VPS4B triggers an inflammatory response in colorectal cancer (2019)
Figure 6

Figure 6. Synthetic lethality between VPS4A and VPS4B induces release of immunogenic DAMPs and promotes M1 macrophage polarization.

A, B Measurement of ATP (A) and HMGB1 (B) released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture. Data are means of 5 (A) or 4 (B) independent experiments ±SEM. Two-tailed unpaired t-test; **p<0.01, ***p<0.001.

C Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.

D Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody.

E qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.

The exact p-values can be found in the source data for this figure.
Translocation of nutrient transporters to cell membrane via Golgi-bypass in Aspergillus nidulans (2020)
Figure 2

Fig.2. Neosynthesized UapA does not co-localize with Golgi markers.

(A, B) Co-localization analysis of neosynthesized alcAp-UapA-GFP with early (SedV) or late (PHosbp) markers, respectively, tagged with mCherry or mRFP. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization was performed by calculating Pearson's Correlation Coefficient (PCC). One sample t-test was performed to test the significance of differences in PCCs. Biological/technical replicates:2/9 for alcAp-UapA-GFP mCherry-SedV and 2/8 for alcAp-UapA-GFP mRFP-PHosbp. For the definition of the two categories of replicates see Materials and methods. Results of quantification, shown on the middle, suggest that there is no significant overlapping fluorescent signal of UapA with SedV (PCC=0.25±0.09, P<0.0001) or with PHOSBP (PCC=0.34±0.06, P<0.0001), as PCC values close to 0.2-0.3 are also commonly obtained when distinct compartments (e.g. ER and early Golgi or early and late Golgi) are followed with different fluorophores. Scale bars: 2 μm.

(C, D) Co-localization analysis of neosynthesized alcAp-GFP-SynA, used as a conventional cargo that traffics through the Golgi, with early (SedV) or late (PHospb) markers tagged with mCherry or mRFP, respectively. Images show single hyphal cells. For conditions of transcriptional derepression see Materials and methods. Quantification of co-localization and statistical analysis was performed as in (A, B). Biological/technical replicates: 2/10 for each strain. Statistical analysis showed significant colocalization of SynA with the late Golgi marker (PCC = 0.67±0.04, P<0.0001), but not with early Golgi marker (PCC=0.37±0.06, P<0.0001). An explanation for lack of co-localization of SynA with SedV is given in the text. Scale bars: 2 μm. The cartoon at the bottom depicts the localization of early or late markers as established here in several previous studies (see [20,21]).
Translocation of nutrient transporters to cell membrane via Golgi-bypass in Aspergillus nidulans (2020)
Figure 5

Fig.5 UapA translocation to the PM takes place without involvement of conventional post-Golgi machinery.

(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA-GFP, after 6-8 hours of initiation of transcription in strains where the expression of RabE, AP-1σ or ClaH (thiAp-rabE, thiAp-ap1σ or thiAp-claH) has been repressed ab initio (o/n) by addition of thiamine. Notice that when rabE or ap1σ is repressed UapA-GFP still reaches the PM. In contrast, repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network. Scale bars: 5 μm. Quantification: UapA-GFP PM/cytosolic intensity ratios are plotted on the right. Mean ratios for wild-type, thiAp-rabE, thiAp-ap1σ and thiAp-claH are 0.55±0.02, 0.53±0.03, 0.54±0.02 and 0.29±0.02 respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). After 6-8h of transcriptional derepression, UapA-GFP fluorescence to the PM does not change when rabE or ap1σ are repressed. On the other hand, in the absence of ClaH, UapA does not reach the PM, as its fluorescence intensity there is statistically lower (****P<0.0001) in comparison to that of the wild-type strain. Biological/technical replicates: 2/15 for each strain.

(B) In a similar experiment, the apical polarized localization of de novo made SynA, used as a control of conventional secretion, is shown to be abolished in all three strains where RabE, AP-1σ or ClaH are repressed. Scale bars: 5 μm. mCherry-SynA PM/cytosolic intensity ratios are plotted with values being 0.52±0.02 for wild-type, 0.14±0.02 for thiAp-rabE, 0.24±0.02 for thiAp-ap1σ and 0.16±0.02 for thiAp-claH. The statistical evaluation was performed as in (A). There is a significant difference (****P<0.0001) of mCherry-SynA fluorescence in the apical PM membrane between the wild-type and the three strains lacking the post-Golgi proteins. Biological/technical replicates: 2/15 for each strain.

(C) Co-localization analysis and relevant quantification of strains co-expressing de novo made UapA-GFP (120 min after initiation of transcription) with RabE-mRFP, AP-1-mRFP or ClaH-mRFP. Scale bars: 2 μm. Quantification by calculating Pearson's Correlation Coefficient (PCC) shows clear non-colocalization of UapA with all the Post-Golgi markers tested, as confirmed by one sample t-test (PCC=0.31±0.08, 0.29±0.04 or 0.07±0.01, with ****P<0.0001, for RabE, AP-1 or ClaH, respectively). Biological/technical replicates: 3/7 for each strain.
Translocation of nutrient transporters to cell membrane via Golgi-bypass in Aspergillus nidulans (2020)
Figure 6

Fig.6. Actin organization, but not microtubules, are essential for UapA translocation to the PM.

(A) Time course of treatment with the actin polymerization drug latrunculin B for 2, 5, 10, 30 or 50 minutes of a strain expressing neosynthesized UapA-GFP under conditions of derepression compared to an untreated strain included as control (150 min). In all cases latrunculin B was added at 100 min of UapA derepression, so that total time of UapA-GFP expression was 102, 105, 110, 130, or 150 minutes in the different samples. Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min, respectively. Scale bar: 5 μm.

(B) Time course of treatment of strains expressing neosynthesized alcAp-UapA-GFP and mCherry-TubA with the anti-microtubule drug benomyl for 5, 55, or 95 minutes. In all cases benomyl was added at 95 min of UapA derepression, so that total time of UapA-GFP expression was 100, 150 or 190 minutes Benomyl abolished the thread-like appearance microtubules in all samples added, evident by the diffuse cytoplasmic signal of mCherry-TubA. Notice that UapA reaches normally the PM (at 190 min of derepression), similarly to the untreated control (right panel). Scale bar: 5 μm.

(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm.

(D) Effect of latrunculin B or benomyl on COPII vesicle formation, followed through the subcellular localization Sec24-GFP. Notice that 40 min of latrunculin B led to loss of the wild-type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm. In contrast, benomyl treatment did not affect the wild-type punctuate localization of Sec24. Scale bar: 5 μm.
Translocation of nutrient transporters to cell membrane via Golgi-bypass in Aspergillus nidulans (2020)
Figure 7

Fig.7. The t-SNARE SsoA is essential for UapA translocation to the PM.

(A) Subcellular localization of de novo made alcAp-GFP-SsoA in the PM in a single hypha. Notice the ER-like membranous network (white arrows) and several cytosolic puncta labelled before SsoA reaches eventually the PM. Scale bar: 5 μm.

(B) Subcellular localization of neosynthesized UapA-GFP (8 hours after transcriptional depression) while ssoA transcription is ab initio repressed. Notice the localization of UapA in the ER-membranous network, often at very close proximity with the PM. Scale bar: 5 μm. In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when ssoA is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.30±0.02 respectively. There is statistically lower fluorescence intensity to the PM (****P<0.0001) when SsoA is absent from the cell, as confirmed by an unpaired t-test. Biological/technical replicates: 2/15 for each condition.
Translocation of nutrient transporters to cell membrane via Golgi-bypass in Aspergillus nidulans (2020)
Figure 8

Fig.8. Other transporters follow the trafficking route of UapA.

(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm.

(B) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters (see text for details) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated. Notice that addition of Benomyl, has no effect on translocation of AzgA and FurA to the PM, whereas Latrunculin B abolishes their PM localization, as seen also for UapA. Scale bar: 5 μm.
HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma induced apoptosis (2021)
Figure 2

Figure 2: HOIP-deficient melanoma cells exhibit increased sensitivity to combined TNF and IFN-γ

(A) Representative flow cytometry plots (left) and pooled data (right) of competition assays with sgControl and sgRnf31 B16-F10 ova tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.

(B) CRISPR/Cas9 screen showing depleted B16-F10 genes after one week culture with TNF + IFN-γ (both 10 ng/mL); blue genes indicate overall genes with p < 0.001, black genes indicate genes of interest with p < 0.05. Significance assessed using MAGeCK test.

(C) Individual log2-transformed sgRNA count values from (B) for Rnf31 (left) and Rbck1 (right); significance assessed using MAGeCK test.

(D) GO term analysis showing enriched biological processes for the top depleted genes from (B).

(E) TNF/IFN-γ and NK cell CRISPR/Cas9 screen comparison showing shared depleted genes; indicated genes are p < 0.01 and filtered for 3/4 affected sgRNA in both screens.

(F) TNF/IFN-γ and OT-I CRISPR/Cas9 screen comparison showing shared depleted genes; indicated genes are p < 0.001 and filtered for 3/4 affected sgRNA in both screens.

(G) Western blot analysis on whole cell lysates showing HOIP expression for sgControl and sgRNF31 A375 tumor cells created using CRISPR/Cas9; β-actin was used as a loading control.

(H) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of sgControl or sgRNF31 A375 tumor cells after culture with the indicated recombinant cytokine(s) (10 ng/mL) for 48 hours; n.s.: no significance, *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.

Data information: See also Figure EV2 and Dataset EV1
HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma induced apoptosis (2021)
Figure 3

Figure 3: Combined TNF and IFN-γ induces transcription-dependent apoptosis in HOIP-deficient cells that requires both intrinsic and extrinsic apoptotic machinery

(A) PI staining of sgControl or sgRnf31 B16-F10 tumor cells treated with combined TNF/IFN-γ (both 10 ng/mL) for 24 hours in the absence and presence of QVD-OPh (10 μM); n.s.: no significance, ***p < 0.001, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.

(B) Western blot analysis of the indicated proteins on whole cell lysates for sgControl or sgRnf31 B16-F10 tumor cells cultured with the indicated cytokine(s) (10 ng/mL for all) for 0, 3, 6, or 9 hours; β-actin was used as a loading control.

(C) Schematic outlining experimental design of CRISPR/Cas9 screening for sgRnf31 B16-F10 resistance to killing by combined TNF/IFN-γ (both 10 ng/ml).

(D) CRISPR/Cas9 screen showing enriched genes from sgRnf31 B16-F10 tumor cells after successive treatment with combined TNF/IFN-γ, as described in (C); red genes indicate overall genes with p < 0.0001. Significance assessed using MAGeCK test.

(E) Individual log2-transformed sgRNA count values from (D) for genes associated with classical IFN-γ (top) and TNF (bottom) signaling; significance assessed using MAGeCK test.

(F) GO term analysis showing enriched biological processes for the top enriched genes from (D); the top 10 terms are displayed.

(G) PI staining of sgControl, sgRnf31, sgRnf31/Stat1, sgRnf31/Casp8, sgRnf31/Bid, or sgRnf31/Rela B16-F10 tumor cells treated with combined TNF/IFN-γ for 24 hours; *p < 0.05, ****p < 0.0001, asterisk(s) indicated group vs TNF/IFN-γ-treated sgRnf31 group, Unpaired t-test for n > 3 biological replicates. Pooled data are represented as mean ± SEM.

Data information: See also Figure EV3 and Dataset EV2
HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma induced apoptosis (2021)
Figure 4

Figure 4: Pharmacological inhibition of HOIP sensitizes tumor cells to combined TNF and IFN-γ induced apoptosis in vitro

(A) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with recombinant TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); n.s.: no significance, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.

(B) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with supernatant (Sup) (derived from a previous overnight OT-I/B16-F10 ova co-culture at the indicated E:T ratio for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of Sup); *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.

(C) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental A375 tumor cells left untreated or treated with TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); *p < 0.05, **p < 0.01, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.

Data information: See also Figure EV4
HOIP limits anti-tumor immunity by protecting against combined TNF and IFN-gamma induced apoptosis (2021)
Figure 5

Figure 5: HOIP-deficient tumors exhibit enhanced OT-I-mediated control in vivo

(A) Experimental design of in vivo model with subcutaneous B16-F10 ova tumors treated with adoptive OT-I therapy.

(B) Individual (left) and average (right) in vivo growth of subcutaneous wild-type or sgRnf31 B16-F10 ova tumors left untreated or treated with adoptively transferred OT-I CD8+ T cells (on days indicated by black arrows); *p < 0.05, **p < 0.01, Unpaired t-test for OT-I-treated wild-type vs OT-I-treated sgRnf31 with n = 5/6 mice per group. Pooled data are represented as mean ± SEM.

(C) Overall survival of mice from (B); *p < 0.05, **p < 0.01, Log-rank test.

Data information: See also Figure EV5

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