EMBO/SourceData huggingface dataset:
Figure 6A: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng/mL TexasRed-EGF (TxRed-EGF) for 30 min. Scale bar: 10 μm. B: Quantification of TxRed-EGF puncta per cell, relative to the number taken up by control cells (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.C: Endocytosis rate of wild type (WT) EGFR was assayed in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells by cell surface biotinylation and one application of 2 ng/mL EGF for 15 min. Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.D: Plasma membrane recycling rate of WT EGFR in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng/mL EGF treatments for 15 min Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.E: Glial shNf-1/shTp53 control and sgAtg7 cells stably overexpressing mCherry-Rab11 were serum starved 4 hr then stimulated with 20 ng/mL EGF for 15 min then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 μm. F: Quantification of Pearson's colocalisation coefficient between mCherry-Rab11 and EGFR (in E). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.
Figure 7Glial shNf-1/shTp53 sgAtg7 (A) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.Glial shNf-1/shTp53 sgAtg16l1 (B) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.Glial shNf-1/shTp53 sgGal8 (C) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.D: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 2 ng/mL EGF for the indicated times before analysis of EGFR signalling pathway activity by western blotting. E: Phosphorylated:total protein ratios for EGFR and AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation (100%) (for EGFR: t=5 min in control cells and for AKT: t=7 min in control cells) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.F: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr with or without 20 ng/mL EGF 30 min stimulation before subcellular fractionation and analysis by western blotting.G: Radar chart indicating the relative levels of pAKT (pink line), pEGFR (light blue line), or total EGFR levels (dark blue line), in autophagy-deficient cell lines relative to their control counterparts. The following knockouts were introduced: glial cells (sgAtg7), HT29 (crATG7), MCF-7 (sgATG5), A549 (sgATG5), U2OS (sgATG5), and MEFs (Atg16l1-/-).H: Control or sgAtg7 glial shNf-1/shTp53 cells were serum starved for 24 hr in the presence or absence of 20 ng/mL EGF. Quantification of the percentage of propidium iodide (PI)-positive cells is shown. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.
Figure 1(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).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.
Figure 225HC inhibits membrane fusion mediated by SARS-CoV-2 S (F) 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding SARS-CoV-2 S (F) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.25HC inhibits membrane fusion mediated by , SARS-CoV S (G, left), and MERS-CoV S (G, right). 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding , SARS-CoV S (G, left), and MERS-CoV S (G, right) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. Scale bar, 50 μm. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.
Figure 3(B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy.(D) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC-treated cells. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16h. Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h. After further incubation with ALOD4, cells were washed with PBS twice and lysed. The amount of bound ALOD4 was examined by western blotting.Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. Entry of SARS-CoV-2 pseudovirus (E and F) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus, and MERS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. , SARS-CoV pseudovirus (G), and MERS-CoV pseudovirus (H) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.
Figure 4(C) SZ58-035 rescues surface accessible cholesterol in 25HC-treated cells. Calu-3 cells were treated with vehicle or 40 µM SZ58-035 for 16 h, prior to treatment with ethanol or 2.5 µM 25HC for 1h. Cells were then incubated with ALOD4 for 30 min. The amount of bound ALOD4 was examined by western blotting.
Figure 1A, Quantification of Nox4 mRNA levels (A) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group.B Quantification of Nox4 protein levels (B) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum-replete cells (15% serum). n=4 cell preparations/group. A representative immunoblot for Nox4 and tubulin as a loading control is shown in panel B.C Cell death after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA (Ad.shNox4) as compared to cells infected with a control vector (Ad.Ctl). n=6/group. The representative immunoblot shows reduction in Nox4 protein levels after shRNA-mediated knockdown.D Lactate dehydrogenase (LDH) activity in cell supernatants. Staurosporine (Stauro, 1 μmol/L) was used to induce cell death by apoptosis. n=4 cell preparations/group.E Levels of high mobility group box1 protein (HMGB1) in cell supernatants. n=3/group. A representative immunoblot is shown at the top.F Protein levels of cytochrome c in the cytoplasmic fractions of cardiomyocytes. n=4/group. A representative immunoblot is shown at the top. The cellular membrane fraction (Mem) containing mitochondria was used as a positive control.G Percentage of Nox4-depleted and control cells with depolarized mitochondria, quantified by flow cytometry. n=3 cell preparations/group (100,000 cells per experiment).H Effect of cyclosporin A (CsA, 1 μmol/L) on the percentage of cells with depolarized mitochondria. n=6-8/group.I Effect of cyclosporin A (CsA, 1 μmol/L) on cell death in serum-starved cardiomyocytes. n=4/group.J Cell death in serum starved Nox4 knockout MEFs (Nox4KO) and wild-type MEFs (WT). Cat = PEG-catalase (500 U/mL). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4P437H (Mut). n=6-12/group.K HMGB1 levels in the supernatants of serum-starved MEFs in similar experiments to those in (J). n=4/group. A representative immunoblot is shown to the top.L Percentage of MEFs with depolarized mitochondria. n=6/group.
Figure 2A Basal mitochondrial calcium levels assessed in serum-starved rat cardiomyocytes using a mitochondrial-targeted cameleon CFP/YFP FRET probe. Nox4 was depleted with silencing RNAs (siRNAs) or cells were transfected with a control scrambled siRNA (siScr). Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). The mean changes in Nox4-depleted cells as compared to scrambled control (dotted line) at the bottom. The representative immunoblot shows depletion of Nox4 protein levels. Scale bars: 10 µm. n=3/group (with >50 cells per individual experiment).B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation. Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). Mean data at the bottom. Scale bars: 10 µm. n=3/group (with >100 cells per individual experiment).Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, C) n=3/group (with >30 cells per individual experiment).D Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, D). n=3/group (with >30 cells per individual experiment).E, Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, E) n=3/group (with >30 cells per individual experiment).F Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, F). n=3/group (with >30 cells per individual experiment).G Peak increase in mitochondrial calcium levels response in response to histamine (100 μmol/L) in WT MEFs with or without treatment with PEG-catalase (Cat), and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant (Mut). n=3/group (with >30 cells per individual experiment).H Representative time-course of histamine-induced changes in mitochondrial calcium for the experiments shown in (I).
Figure 4A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue) and yellow dots corresponding to co-localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP3R/Nox4, and FACL4/InsP3R. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).C Simplified photomicrographs of proximity ligation staining in hiPSC-CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment).
Figure 5A Immunoblotting of subcellular fractions from serum-starved cardiomyoblasts for InsP3R, Akt and PP2a, as in Fig 3A.B Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5-60%) and immunoblotting for relevant proteins. Fraction size: 200 µL. Representative of 3 independent experiments.C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum-starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG-catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant (Mut). n=3/group.D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS-insensitive SypHer probes was used to correct for any pH-induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n=5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 µm.
Figure 6B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment.C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment.D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group.E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group.F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Mean data shown to the right. n=3/group.G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.
Figure 1A Analysis of MNase-digested chromatin DNA fragments from wild-type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells.B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.C Calculation of nucleosome fuzziness as the standard deviation of nucleosome (or tag) positions. See also Appendix Fig S1.
Figure 2B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2-H2AΔ14-H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A-C that are related by two perpendicular non-crystallographic dyads ().C Side-view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.D View of the yNap1-H2A-H2B protomer complex with labeling of secondary structure elements.
Figure 3A Overview of the yNap1c2-H2AΔ14-H2BΔ28 complex.B Residues R17, R20, R29, R32, R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201, D205 and E310.C Residues K75, R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332, D333 and E339 in α8 of yNap1.D In vitro GST-pulldown assay showing the role of residues in the HBR interfaces in H2A-H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input.E Disruption of the Nap1-histone interaction by H2A mutation. Bottom panel: Inputs of the coexpressed proteins (soluble extract) and top panel the material bound to the glutathione affinity column were analyzed by SDS-PAGE and Coomassie staining.F Controls showing input (bottom) and bound (top) fractions. See also Fig EV2.
Figure 5A EM analysis of the yNap1c2-H2AΔ14-H2BΔ28 complex. Representative rawimage of negatively-stained particles.B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row).C From left to right: Crystal structure of the (yNap1c2-H2A-H2B)6 complex in two different orientations (brown); 60 Å-filtered structure of the complex (magenta); 3D reconstruction of the complex from negatively stained particles (green) and fit of the crystal structure into the EM map.D-I Mass spectra of yNap1 variants in the absence and presence of H2A-H2B. yNap1 in complex in the absence (D) or presence of H2A-H2B (E). yNap1c in the absence (F) or bound to H2A-H2B (G). The oligomerization mutant yNap1_IF1 alone (H) or in complex with H2A_IF2Δ14-H2BΔ28 (I). yNap1 is shown in green circles and H2A-H2B as red and blue asterisks.
Figure 6A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488- (left) or AF647-labeled DNA (right). 31 nM - 4 µM of yNap1-H2A-H2B (lanes 6-13) were incubated with a mixture of 0.8 µM AF488-labeled H3-H4-DNA tetrasome complexes and 0.8 µM AF647-labeled DNA (lane 5). Lanes 1-3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A-H2B obtained by salt deposition onto AF647-labeled DNA.B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.C Analysis of MNase-digested chromatin DNA fragments from wild-type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker.
Figure 2a) Kaplan-Meier to first fever (≥38°C) episode within 24 hours of receiving vaccines administered at 4 months of age; control n=88 and 4CMenB n=89.b) Principal component (PC) analysis of RNA-sequencing data (14837 genes, n= 253) from all study time points. The lines connect participant's pre- and post-vaccination samples. The ellipses are the two-dimensional (PC1 and PC2) 95% confidence intervals for each study time point. A contribution plot (top right) displays the genes contributing most to PC1/PC2, i.e. 0.15 implies 0.15% contribution of that variable to the principal components displayed.c) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA-sequencing data, with median and interquartile range. P-values were determined from a two sample Wilcoxon rank sum test. The number of individuals in each group is display in the x-axis.d) Spearman's rank correlation between neutrophil counts measure by full blood counts (n= 142) and those estimated by CIBERSORTx, using the LM22 signature matrix.
Figure 3a) Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination (4 months of age) 4 hours post-vaccination (719 DEGs, n=28). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). The top 10 genes, ranked by FDR, are labelled.Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination b) Same as (a) but 24 hours post-vaccination (5553 DEGs, n=31)Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination c) Same as (a) but 3 days post-vaccination (159 DEGs, n=30)Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination d) Same as (a) but 7 days post-vaccination (6 DEGs, n=36).