EMBO/soda-nel huggingface dataset:
The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection (2022)
Figure 1

Figure 1: Characterisation of phagosomal ubiquitylation.

(A) Intraphagosomal proteolysis assay shows that IFN-γ activation of macrophages reduces phagosomal maturation. Shaded area represents SEM (six technical replicates; representative graph of three independent experiments).

(B) Workflow of phagosome isolation.

(C-E) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate (TCL) and further increases by IFN-γ activation (C, E). Equal protein amounts were loaded for phagosomal and TCL samples. Vimentin serves as loading control; EEA1 as purity marker. (D) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome (30 min pulse, 0, 30, and 180 min chase, respectively). LAMP1 serves as purity marker. Representative images of three replicates.

(F) Ubiquitin AQUA PRM assay for phagosomes from RAW264.7 cells shows abundance of atypical chains and increases for K6, K11, K48 and K63 chains on phagosomes in response to IFN-γ activation. As the experiment is complex and has inherent variability, five independent experiments of three biological replicates were combined. M1 represents linear ubiquitin chains. M1= linear ubiquitin chains. Error bars represent SEM of five biological replicates.

(G) Ubiquitin AQUA PRM assay of total cell lysates of RAW264.7 cells. Error bars represent SEM of three technical replicates in three independent experiments.

(H) Ubiquitin Chain Restriction (UbiCRest) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs. UT = untreated.

(I) Representative immunofluorescence micrograph showing that K63 polyubiquitin (in green) localises around the phagosome defined by Rab7 staining (in red). Bead size (in white): 3 μm. To account for people with red-green colour-blindness, we have added a magenta/cyan/yellow version in Appendix Figure S2. Scale bar is 2 µm.

Data information: *, p-value < 0.05; **, p-value < 0.01 by paired two-tailed Student's t-test. Relative mobilities of reference proteins (masses in kDa) are shown on the right of each blot.
The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection (2022)
Figure 3

Figure 3: Ubiquitin E3 ligase RNF115 is enriched on phagosomes of IFN-γ activated macrophages and loss of RNF115 affects several phagosomal protein compositions.

(A) Proteomics intensity levels of selected phagosomal E3 ligases enriched upon IFN-γ activation. Missing values for RNF115 and Herc6 in resting macrophages have been added by imputation. Standard deviation bars represent SD of three biological replicates.

(B) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264.7 cells and its increased abundance in response to IFN-γ activation. Rab7 serves as purity control, iNOS as activation control and vimentin as loading control. Representative image of three replicates. Relative mobilities of reference proteins (masses in kDa) are shown on the right of each blot.

(C) Representative immunofluorescence micrograph showing RNF115 co-staining with LAMP1 around the phagosome in murine BMDMs. Scale bar equals 5 µm.

(D) Volcano plot of proteomics data of resting WT and RNF115 KO BMDM phagosomes. Selected proteins are highlighted.

(E) Gene Ontology (GO) analysis of enriched and depleted proteins from (D).

(F) Proteomics intensity data of selected protein complexes derived from (D). Error bars represent of three biological replicates.

Data information: n.d., not detected; *, p-value < 0.05; **, p-value < 0.01 by unpaired two-tailed Student's t-test.
The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection (2022)
Figure 4

Figure 4: Loss of RNF115 increases phagosomal proteolysis and enhances innate immune responses to intraphagosomal bacteria.

(A-B) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells.

(C) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes, shows that RNF115 ligase activity is required for the increase of phagosome maturation. Shaded areas represent SEM.

Data information: six technical replicates; representative graphs of three independent experiments.
The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection (2022)
Figure 5

Figure 5: Pull-down of ubiquitylated proteins from phagosomes from WT and RNF115 KO macrophages indicates RNF115 ubiquitylates various proteins involved in immune responses and vesicle trafficking and may affect formation of the VAMP8/STX7 SNARE complex.

(A) Volcano plot of proteomics experiment of Gly-Gly peptides from phagosomal extracts of WT and RNF115 KO macrophages. Selected proteins are highlighted.

(B) Network representation (from STRING v11) (Szklarczyk et al., 2019) of selected proteins with reduced ubiquitylated peptide abundance in response to loss of RNF115, i.e. potential phagosomal substrates of RNF115.

(C) Immunofluorescence micrographs of VAMP8 (green) and Syntaxin-7 (STX7; red) show strong colocation around 3 µm silica bead phagosomes (white) in BMA macrophages. Nuclei are stained with Dapi. Scale bar is 5 µm. To account for people with red-green colour-blindness, we have added a magenta/cyan/yellow version in Appendix Figure S5.

(D) Colocation of VAMP8 (green) and STX7 (red) on individual phagosomes between WT and RNF115 KO cells represented by intersection, Manders' M1 and overlap coefficients. Data is represented as a box and whisker plot of total values across biological duplicate experiments, where the whiskers represent the minimum to maximum values and the central band indicates the median. Error bars represent SEM of two technical replicates of two biological replicates. *, p-value < 0.05; by unpaired two-tailed Student's t-test.

(E) Structure of VAMP8 and STX7 complex onto which a single ubiquitin molecule on VAMP8 K59 was modelled. Inlay: ubiquitylation of K59 disrupts a hydrogen bond between VAMP8 K59 and STX7 Q219.
The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection (2022)
Figure 7

Figure 7: Loss of RNF115 increases inflammatory response in vitro but reduces tissue damage to S. aureus infection in vivo.

(A) Infection of WT and RNF115 KO BMA cells with S. aureus for 6 h. Output/input values correspond to the number of bacteria inside the cell recovered at the specific time point normalised over the number of bacteria added to the cells at the start of the infection. UI, uninfected cells. Standard deviation bars represent SD of four biological replicates.

(B) ELISA data show that loss of RNF115 increases TNF-α and IL6 secretion after 6 h of infection with S. aureus and in uninfected (UI) cells. Error bars represent SEM (n=3 biological replicates, representative graph from three independent experiments). Statistical analysis was don through paired two-tailed Student's t-test.

(C) WT and RNF115 mice were infected with S. aureus and after 48 h CFUs were counted in liver and spleen. Error bars represent SEM of six biological replicates. There was no significant difference between WT and RNF115 KO mice.

(D) Box and whisker plot represents the quantification of the percentage (%) of the hepatic lesion area in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Haematoxylin and eosin (H&E)-stained liver sections of one representative animal per genotype are shown on the right, dotted line depicts the damaged area. Groups were compared using an unpaired two-tailed t-test. The statistical significance of the comparisons is indicated as follows: Each dot represents an individual animal.

(E-F) Box and whisker plots represent the average cell counts per high power field (HPF) of CD3 positive T-cells (E), and CD68 positive macrophages (F) in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Representative immunohistochemistry sections are shown on the right. Groups were compared using an unpaired two-tailed t-test. The statistical significance of the comparisons is indicated as follows: **, P < 0.01. Each dot represents an individual animal.

Data information: *=p<0.05; **, P < 0.01; ***=p<0.001
Virus induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade (2019)
Figure 1

Figure 1. BTV impairs IFN-I and -II signaling

(A and B) (A) ISRE-luciferase reporter assay in 293T cells or (B) GAS-luciferase reporter assay in Vero cells infected with BTV-8 or mock-infected (Control) and treated with (A) 1000 U/ml of universal-IFN, or (B) 5 ng/ml of IFN-γ. Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented. * p<0.05; *** p< 0.001, one-way ANOVA

(C and D) qRT-PCR analysis of ISGs induction in mock- or BTV-8-infected (C) 293T or (D) Vero cells after (C) type I or (D) type II IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented. * p<0.05; *** p< 0.001, one-way ANOVA.
Virus induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade (2019)
Figure 2

Figure 2. BTV prevents STAT1 phosphorylation and nuclear translocation and reduces STAT2 levels. (A) Immunoblot of Vero cells infected with BTV-8 for 16 h, and probed for total and phosphorylated STAT1/2 levels.

(B) Vero cells infected with BTV-8 were fixed at 16hpi, and stained with anti-BTV-VP7 antibody.

(C and D) STAT1 phosphorylation in IFN-I-treated Vero cells infected with BTV-8 detected by (C) flow cytometry and (D) confocal microscopy (Scale bar = 30 µm). Means ± SD of P-STAT1 mean fluorescence intensity (MFI) in 3 experiments are shown. * p< 0.05, one-way ANOVA (Mock + IFN vs infection).

(E and F) STAT2 expression in BTV-8-infected Vero cells detected by (E) flow cytometry and (F) confocal microscopy (Scale bar = 30 µm). Means ± SD of STAT2 MFI in 3 experiments are shown. ** p< 0.01; *** p< 0.001, one-way ANOVA (Mock vs infection).
Virus induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade (2019)
Figure 3

Figure 3. BTV infection induces STAT2 degradation through the lysosome/autophagy pathway

(A-C) Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), or bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. (A) Representative flow cytometry histogram of BTV-VP7 expression in inhibitor-treated cells. (B and C) Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.

(D) Immunofluorescence of BTV-8 or mock-infected Vero cells (treated for the last 6h with BAF-A1) stained for BTV proteins, STAT2 and p62. Inset shows detail of STAT2 and p62 colocalization. Nuclei were counterstained with DAPI. Arrowheads in inset indicate p62 and STAT2 colocalization. Scale bar = 20 μm. Inset scale bar = 3 μm.
Virus induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade (2019)
Figure 4

Figure 4. BTV-NS3 and NS4 contribute to IFN signaling antagonism

(A and B) (A) ISRE-luciferase reporter assays in 293T cells stimulated with IFN-U and (B) GAS-luciferase reporter assays in Vero cells stimulated with IFN-γ co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown. *** p< 0.001, one-way ANOVA.

(C) Immunoblot of 293T cells transfected with BTV -NS2, -NS3, -NS4 (FLAG) plasmids and NiV-V (HA) and WNV-NS5 (HA) used as control for STAT1 phosphorylation inhibition and DENV-NS5 (HA) used as control for STAT2 degradation.

(D) Three independent immunoblot experiments as in (C) were quantified for STAT1, P-STAT1, STAT2 and P-STAT2. Mean ± SD normalized to GAPDH and non-transfected cells. ** p< 0.01; *** p< 0.001, one-way ANOVA (Mock vs transfected cells).

(E) Immunoprecipitation (IP) for STAT1/2 binding using 293T transfected with BTV -NS2, -NS3, or -NS4 plasmids. NiV-V used as positive control for STAT1 and STAT2 binding, DENV-NS5 used as positive control for STAT2 binding. Cells were treated (or left untreated) with 1000 U/ml universal-IFN for the final 16 h. Immunoblots of WCE and IP were probed (IB) for STAT1, STAT2, HA (NiV V, DENV NS5), FLAG (NS2, NS3, NS4) and GAPDH.

(F and G) (F) STAT1 phosphorylation and (G) STAT2 nuclear translocation in BTV-NS3-transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment. (Scale bar = 20 µm). Arrowheads in merge images indicate examples of transfected cells.
Virus induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade (2019)
Figure 5

Figure 5. BTV-NS3 mediates STAT2 degradation through an autophagy/lysosome pathway

(A) Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH.

(B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression.

(E and F) 293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for (E) Atg7 or (F) Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7/Beclin-1; FLAG-tagged NS3; LC3 and GAPDH.

(G) Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells.

(H) Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH.

(I and J) Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.
Virus induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade (2019)
Figure 6

Figure 6. BTV-NS3 protein ubiquitination by K63 Ub chains is critical to its IFN-I antagonistic effects

(A) WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression.

(B) Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm).

(C) Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub.

(D) FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression.

(E) WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression.

(F) ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown. *** p< 0.001, one-way ANOVA.

(G) FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.
CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer (2018)
Figure 1

Fig 1: CEP55 regulates human breast cancer cell survival.

(A, B) Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines (n=25) and in progression series of MCF10A lines (MCF10AT and MCF10CA), respectively. Tubulin and COX-IV served as loading controls.

(C) A panel of selected breast cancer and near normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean± SEM of three independent experiments.

(D) Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA-MB-231 cells. Two isogenic lines (sh#2 and sh#8) were obtained using two different shRNA sequences as described in method section. COX-IV served as loading control.

(E) Effect of CEP55 knockdown on cell proliferation in MDA-MB-231 cells assessed using the IncuCyte ZOOM® live cell imager (phase-only processing module). The percentage of cell confluence was determined using an IncuCyte mask analyzer. Graph represents the mean±SEM of three independent experiments.

(F) Representative images of colony forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA-MB-231 cells.

(G) Six-week-old female NOD/SCID cohorts of mice were injected in the 4th inguinal mammary fat-pad with the control and CEP55 knockdown cells. Growth rate (area, mm2) of the tumors was measured using digital calliper. Differences in growth were determined using Student's t test. Graph represents the mean tumor area±SEM, n=6 mice/group.

(H) Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag-CEP55 expression construct and two isogenic lines were obtained (CEP55-#16 and CEP55-#17) with its respective empty vector (EV) transfected parental cells. COX-IV served as a loading control.

(I) Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM® live cell imager as described in panel E. Graph represents the mean±SEM of two independent experiments.

(J) Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector (EV) and CEP55-overexpressing (CEP55#17 and CEP55#18) lines.

(K) Representative images of single plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: Cytokeratin-19; Green: F-actin stained by Phallodin and Blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH.
CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer (2018)
Figure 2

Fig 2: CEP55 regulates genomic instability and aneuploidy.

(A) Polyploidy analysis (>4N DNA contents) determined using FACS in control and CEP55 knockdown MDA-MB-231 cells (left panel). Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4.0 software (right panel). Yellow peaks represent subpopulation of polyploidy/aneuploidy. Graph represents the mean±SEM of three independent experiments.

(B) Analysis of chromosome number in control and CEP55 knockdown MDA-MB-231 cells (sh#8) by Giemsa staining of metaphase spreads (left panel). Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy (right panel). n= number of metaphase spreads counted (mean ±95% confidence interval of two independent experiments).

(C) Representative SNP array plots showing chromosomal alterations following CEP55-knockdown in MDA-MB-231 cells. BAF is B allele frequency, value between 0 and 1 and represents the proportion contributed by one SNP allele (B) to the total copy number: BAF is an estimate of NB/(NA+ NB), where NA and NB are the number of A and B alleles, respectively.

(D) Percentage of genome altered evaluated using amplified copies (6-8 copies across genome) in both parental and CEP55 knockdown cells determined using data from SNP arrays. Graph represents the mean±SEM of two independent experiments.

(E) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells. Red box indicates an extra chromosome 20 in CEP55-overexpressing cells.

(F) Correlation between average copy number alterations of genome (CNAs)/tumor and CEP55 mRNA expression in the METABRIC dataset, n=997 patients.

(G) Kaplan-Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival. Cases were subdivided into tertiles. Log-rank (Mantel-Cox) test, P< 0.0001.
CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer (2018)
Figure 3

Fig 3: CEP55 overexpression provides survival advantage during perturbed mitosis

(A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM).

(B, C) Percentage of polyploidy (>4N) following B12536 (5 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.

(D, E) Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with PLK1i or Nocodazole as described in panel B,C. Graph represents the mean±SEM of three independent experiments.

(F) Percentage of sub-G1 fraction following B12536 (10 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown Hs578T cells. Graph represents the mean±SEM of two independent experiments.

(G) Representative cytogram of empty vector (EV) and CEP55-overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 (10 nM).

(H) Percentage of polyploidy following treatment with and without B12536 (10 nM) in empty vector (EV) and CEP55-overepressing MCF10A cells. Graph represents the mean±SEM of three independent experiments.

(I) Corresponding sub-G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H. Graph represents the mean±SEM of three independent experiments.