EMBO/SourceData huggingface dataset:
Figure 4Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.
Figure 5Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT -APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.
Figure 6Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).
Figure 1A 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-FLAGimmunoprecipitation (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.
Figure 2A 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.
Figure 3A 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&amp;amp;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 mice 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-testG 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
Figure 4A 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-testD 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).
Fig. 2molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab.dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.
Fig. 3viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells
Fig. 4usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.
Figure 1A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Figure 2A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Figure 3A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Figure 4A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.
Figure 6A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student's t-test.
Figure 7A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.
Figure 8A. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.E. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.
Figure 1B Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control.C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.E Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.
Figure 2A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10.B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.