EMBO/soda-nel huggingface dataset:
USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma (2016)
Figure 1

Figure 1 - USP9X binds, deubiquitylates and stabilizes XIAP in mitosis

A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.

B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.

C Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.

D Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in (C), but with additional transfection of the FLAG-tagged XIAP single amino acid mutants G188R and G188E. Only mitotic samples are shown. The asterisk denotes ubiquitylated forms of XIAP.

E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAG immunoprecipitation (IP) under denaturing conditions.

F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S. The band in the EV control lane of the anti-V5 panel marks an unspecific band produced by the antibody.

G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated.
USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma (2016)
Figure 2

Figure 2 - USP9X stabilizes XIAP to antagonize mitotic cell death independent of MCL1

A Immunoblot analysis of WT, Xiap-/-, Mcl1-/- or cIap2-/- mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non-relevant mRNA (Ctrl) or against Usp9X mRNA and treated with taxol as specified.

B Two-dimensional cell-cycle analysis (BrdU/PI) of cells described in (A). Sub G1 and G2/M fractions of cells were quantified and averaged with two additional, independent experiments (n=3, ± SD; WT: **, P = 0.0016; ***, P = 0.0004; Mcl1-/-: ***, P = 0.0003; **, P = 0.0017; cIap2-/-: **, P = 0.0043, Student’s t test). Black bars exemplify shRNA Ctrl, white bars shRNA Usp9X samples.

C Immunoblot analysis of HeLa cells transfected with a FLAG-tagged USP9X expression construct or empty vector (EV) and treated with taxol for the indicated times.

D Immunoblot analysis of HeLa cells that were transfected with a FLAG-tagged XIAP expression construct or empty vector and treated as in (C).

E Two dimensional cell-cycle analysis (BrdU/PI) of cells described in (C) and (D). Apoptotic indices represent ratios of sub G1 to G1/S cells and are shown for analyses at the indicated timepoints. **, P = 0.0020; ***, P = 0.0004; Student’s t test.

F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non-relevant mRNA (Ctrl) or against Xiap mRNA and treated with taxol as specified.
USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma (2016)
Figure 3

Figure 3 - USP9X and XIAP promote lymphoma growth and resistance to spindle poisons in vivo

A Immunoblot analysis of sorted Eμ-Myc lymphoma cells that were retrovirally transduced with the indicated IRES-GFP shRNA constructs.

B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A). Prior to injection, cells were sorted for viability (propidium iodide negative) and GFP positivity (sh_Ctrl, black, n = 6; sh_USP9X, gray, n = 5). *P = 0.0293; Mantel-Cox test.

C Kaplan-Meier survival curves of mice that were injected with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A) and treated as in (B) (sh_Ctrl, black, n = 6; sh_XIAP, gray, n = 6). **, P = 0.0020; Mantel-Cox test.

D Histopathology of representative lymph nodes derived from mice shown in (B) to visualize histomorphology by haematoxylin/eosin (H&E), B-cell origin (B220), and spontaneous apoptosis (cleaved Caspase 3) in situ. Scale bars denote 50 μm and 20 μm for insets.

E Histopathology of representative lymph nodes derived from mice shown in (C) which were stained as described in (D).

F Mean tumor burden in animals injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection, assessed by PET imaging (4 animals per group). **, P = 0.0025; ***, P = 0.0003, student’s t-test

G Representative PET images of animals, injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection (pre treatment) and three days following intraperitoneal vincristine administration (post treatment). Bone marrow infiltration, representative for systemic lymphoma manifestation, is indicated by a blue asterisk.

H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden (sh_Ctrl, black, n=6; sh_Usp9X, white, n=8; sh_Xiap, grey, n=8). Within the sh_Usp9X and sh_Xiap groups all animals lived up to control imaging, while two of eight animals of the sh_ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden. These animals had to be excluded from final analysis, suggesting an underestimation of the actual effect. Data was obtained from two independent experiments. ****, P < 0.0001; *, P = 0.0167, student’s t-test
USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma (2016)
Figure 4

Figure 4 - USP9X is overexpressed in DLBCL to promote survival and resistance to anti-tubulin chemotherapeutics via mitotic stabilization of XIAP

A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins.

B FACS analysis (propidium iodide (PI)) of USP9X high and low expressing DLBCL cells shown in (A) to determine cell survival. Cells were treated with taxol and collected at the specified time points. PI negative cells are indicated relative to time point 0 (n=3 ± SD).

C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES-GFP shRNA constructs against USP9X or a non-relevant mRNA, FACS sorted for GFP+ PI- cells and exposed to taxol for the indicated periods of time. The quantification of cleaved caspase 3 (CC3) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA. **, P = 0.0099, student’s t-test

D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time. Three hours before collecting, WP1130 at a concentration of 5μM or DMSO was added as specified.

E FACS analysis (propidium iodide (PI) uptake) of DLBCL cell lines treated with taxol and/or the SMAC mimetic BV6 as indicated. Results displayed are from three independent experiments (n=3 ± SD). ***, P = 0.00085; ***, P = 0.00529; Student’s t test.

F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays (TMA) derived from a patient cohort representing 121 evaluable human CD20+ aggressive B-cell lymphomas. High USP9X expression was identified in 37 cases (31 %) and significantly correlated with high XIAP expression levels (**, P = 0.004; Student’s t test).

G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).
Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and coronaviruses by depleting membrane cholesterol (2020)
Figure 1

TAG

Figure 1. Cholesterol 25-hydroxylase (CH25H) is induced by SARS-CoV-2 and restricts viral infection.

(A) IFNs and ISGs were induced by SARS-CoV-2 infection in lung epithelial cell lines: Calu-3 and A549-ACE2 were infected with SARS-CoV-2 at MOI=2 for 24h; A549 was challenged with IAV at MOI=5 for 9h; A549 was infected with HPIV3 and RSV at MOI=2 for 24h (Blanco-Melo et al., 2020). Color key represents log2(fold change) relative to uninfected control cells. CH25H was highlighted by red asterisk.

(B) Expression of CH25H in heathy donors and COVID-19 patients. The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors, three moderate COVID-19 patients and six severe COVID-19 patients by scRNA-seq analysis (Liao et al., 2020). *p < 0.05, ****p < 0.0001, by Student's t test

(C-E) Characterization of SARS-CoV-2 pseudovirus. (C) SARS-CoV-2, SARS-CoV and VSV pseudovirus (VSVG) were concentrated by ultracentrifuge. Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS-CoV and SARS-CoV-2. VSV Matrix protein served as a loading control for viral particles. Black arrows indicate full length S and cleaved spike (S2 subunit), respectively. (D) SARS-CoV-2 pseudovirus infection in Calu-3 cells with or without treatment of coronavirus fusion peptide EK1. Luciferase activity was measured to quantify the infection. (E) SARS-CoV-2 pseudovirus infection in Calu-3 cells transduced with non-targeting control or ACE2 specific sgRNA. Luciferase activity was measured to quantify the infection. Mean ± SD of n = 3. ***p < 0.001, ****p < 0.0001, by Student's t test.

(F-G) Overexpression of CH25H restricts SARS-CoV-2 entry. Calu-3 cells transduced with lentivirus overexpressing CH25H or empty vector were infected with SARS-CoV-2 pseudovirus encoding Fluc or EGFP and pseudovirus infection was quantified by luciferase assay (F) or visualized by fluorescence microscopy (G). Scale bar, 100 μm. Mean ± SD of n = 3. ****p < 0.0001, by Student's t test.
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 1

Figure 1. Stx17 binds to PGAM5.

Schematic representation of Stx17 and PGAM5.

293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.

HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).

MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.

293T cells were cotransfected with plasmids encoding FLAG-Stx17 WT and the indicated PGAM5-GFP constructs, and analyzed as described in (B).

293T cells were cotransfected with plasmids encoding FLAG-Stx17 or Stx18 and the C-terminally GFP-tagged transmembrane domain of PGAM5（amino acids 1-35）constructs, and analyzed as described in (B).
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 2

Figure 2. PGAM5 is localized at the ER-mitochondria interface.

HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).

HeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test).

HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).

Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Sampled were prepared as described in Materials and methods. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.

HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).

HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 3

Figure 3. Localization of PGAM5 is regulated by Stx17.

HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.

293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.

293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.

HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 4

Figure 4. Mitophagy is inhibited upon depletion of Stx17 or PGAM5.

HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.

HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).

PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 5

Figure 5. PGAM5 depletion abrogates the proximity of GFP-Parkin-associated mitochondria to omegasomes.

GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).

GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).

GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 6

Figure 6. Stx17 is required for the link between PGAM5 and FUNDC1.

HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.

GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.

293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.

GFP-Parkin stably expressing HeLa cells were treated as described in (C) and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.
Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy (2018)
Figure 7

Figure 7. PGAM5 modulates the mitochondrial phenotypes caused by Stx17 inhibition in Drosophila.

A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17-/-; c, Stx17-/- and Stx17 overexpression; d, Stx17-/- and PGAM5 overexpression) of 7-day-old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system : Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *p < 0.05, #p < 0.001 vs. the same class of control (Dunnett's test). n = 50-119 mitochondria from three or four independent samples. Genotypes used were: +/y; Act5c-GAL4/+ (a), +/y; Act5c-GAL4/+; Stx17LL06330/Stx17LL06330 (b), +/y; Act5c-GAL4/UAS-FLAG-Stx17; Stx17LL06330/Stx17LL06330 (c), +/y; Act5c-GAL4/UAS-PGAM5; Stx17LL06330/Stx17LL06330 (d). Scale bars, 200 nm.

B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified as described in (A). *p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).
Mitochondrial localization of GASZ is essential for nuage formation, transposon repression and spermatogenesis (2015)
Figure 1

Figure 1. GASZ contains a mitochondrial localization signal.

A. Predicated MLS localized at the C-terminus of GASZ and is enriched for hydrophobic amino acids that form a helical wheel.

B. Subcellular fractionation and Western blots of 293T cells with ectopically expressed GASZ, NRF1, MVH, or MitoPLD.

C. Localization of GFP fused with wild-type or mutant MLS of GASZ in HeLa cells. Mitochondrial localization was monitored by MitoTracker (Red) staining. Scale bars: 15 m.
Mitochondrial localization of GASZ is essential for nuage formation, transposon repression and spermatogenesis (2015)
Figure 2

Figure 2. Mitochondrial localization of GASZ is essential for its function in male reproduction.

A. IHF of GASZ and Cytochrome C (Cyt C) of testical sections from mice at day 7 postnatal, or MILI and MVH of gonad sections from wild-type embryos or embryos with MLS deletion at 16.5 dpc. Scale bars: 5 m.

B. Western blots of GASZ expression of testes at day 5 postnatal.

C. Gross morphology of testes from adult mice at 3 months postnatal with mutated MLS compared to their wild-type littermates.

D. Histological studies of testis sections from wild-type mice or mice with MLS deletion at different time points after birth. Scale bars: 50 m.

E. EM of testis sections from wild-type neonatal mice or mice with MLS mutation. Red arrows point to IMC among mitochondria.

F. Expression of transposable elements analyzed by real-time RT-PCR on testes from wild-type mice or mice with mutated MLS at various time points after birth. Data were shown as mean (of expression from three biological replicates) ± one s.e.m. n=3 for each genotype. **: p<0.01. *: p<0.05.