EMBO/SourceData huggingface dataset:
Figure 7COVID-19 severity is associated with significant changes in lipoprotein subclasses including high-density lipoprotein subclass-1 (HDL1), HDL4, low-density lipoprotein subclasses (LDL1, LDL4, LDL5), very low-density lipoprotein subclass-5 (VLDL5) and their compositional components (ApoA1, triglycerides, cholesterol). TG: triglycerides; FC: free cholesterol; CE: cholesteryl esters; CH: total cholesterol (i.e., FC + CE); PL: total phospholipids; A1: ApoA1; A2: ApoA2; L1TG: TG in LDL1; L1TG%: percentages of L1TG in total lipids of LDL1; L1%: percentage of LDL1 in all LDL; L-TG%: percentages of L-TG(TG in LDL) in total lipids of LDL; V5FC%, V5CE%: percentages of V5FC(FC in VLDL5) and V5CE(CE in VLDL5) in total lipids of VLDL5; L5CE%, L5CH%: percentages of L5CE(CE in LDL5) and L5CH(CH in LDL5) in total lipids of LDL5; H1FC%: percentages of H1FC (FC in HDL1) in total lipids of HDL1; H-A2: ApoA2 in both HDL and nascent HDL; H4A1, H4A2, H4CE, H4CH, H4FC, H4PL: ApoA1, ApoA2, CE, CH, FC and PL in HDL4;Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID-19 severity. Data were represented as means ± SD and differences between groups were estimated using a Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Control, n=12; mild, n=29; severe, n=17; discharge, n=16; sLDLR: soluble low-density lipoprotein receptor; LCAT: lecithin-cholesterol acyltransferase; CEPT: cholesteryl-ester transfer protein.
Figure 1TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation), from three independent experiments.TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.(B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05).
Figure 2TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(B) (Left panel) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control.TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Figure 3TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments.(B) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(C) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA, and 48 h later exposed to nutrient starvation for 5 h. Actin expression was examined as a loading control.(D) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or DRAM siRNA1/2. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments; * represents significant difference from starved control conditions (P<0.05); @ represents significant difference from untreated control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Figure 4(A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation) from three independent experiments. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments.(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Figure 5(A) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA.(B) RT-PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA.(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h.(D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Figure 6(A) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) were left untreated or exposed to nutrient starvation for 6 h, with or without treatment with Z-VAD-FMK for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation of the mean from three independent experiments.(B) Apoptosis in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3), as measured by cells with a sub-G1 DNA content. Cells were transfected with either scrambled, TIGAR siRNA1 or TIGAR siRNA2. Cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h.(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Figure 1(G) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group (numbered 1-4) was detected by anti-IgG secondary antibody .
Figure 3Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm.Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).
Figure 8(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. summary data for CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.
Figure 1B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3). C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4). D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively. Da
Figure 3A-H. SEM images of an opened embryo (A), the ExE cavity wall (B) where blood islands are indicated by white arrow, the allantois surface (C-C'), an opened allantois (D) and its zooms (E-E') where filaments are indicated by white arrows, the epiblast-derived side of the amnion (F), the mesoderm-derived side of the amnion (G) and its zoom (G').
Figure 4B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm.Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E) , over embryonic region length.I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.
Figure 5F. Dot plot of specific gene expression in amnion clusters.G. Immunostaining for Cdx2 (n=20). H. Immunostaining for Nrp1 (n=8) and Col1a1 (n=7), primarily expressed in Amnion Mesenchyme. I. Immunostaining for Epcam (n=12), Cldn7 (n=7), and Lrp2 (n=6), primarily expressed in Amnion Ectoderm.J-K. Sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Nrp1 (K) (n=8 and 2, respectively), and Lrp2 (L) (n=3 and 2).
Figure 6D. Dot plot of specific gene expression in chorion clusters.E. Immunostaining for Cdx2 (n=20). F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm. G. Immunostaining for Cebpb, detectable in most cells with variable intensity. H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme. I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3). Data
Figure 7D. Dot plot of specific gene expression in allantois clusters.F. Immunostaining for Ifitm3 (n=7) (Allantois Base). G. Immunostaining for Cdh11 (n=9), Stard8 (n=4) and Nrp1 (n=8) (Allantois Cortex). H. Immunostaining for Pitx1 (n=4) (Allantois Mesenchyme). I. Hhex-GFP (n=5) and immunostaining for Kdr (n=8) (Endothelial Progenitors). J. Immunostaining for Runx1 (n=3) (Erythroid Progenitors). K-L.K-L. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Cdh11 (Top) and Krt8 (Bottom) (K, n=8 and 4, respectively) and Ifitm3 (L, n=2 and 2). Data information: White arrows point toward signal in allantois. Sagittal sections with anterior to the left.
Figure 1(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control.(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.
Figure 2(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.(B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.(C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.(D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm.(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.(F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group.(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.
Figure 3(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.(B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm.(D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting.(E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay.(F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies.(G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.
Figure 4(B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed.(C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting.(D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm.(E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting.(F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. .(H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.