EMBO/soda-nel huggingface dataset:
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 3

Figure 3 NAT10 stabilizes p53 by counteracting Mdm2 action

A U2OS cells were transfected with the indicated siRNAs. Seventy-two hours later, cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies.

B U2OS cells were transfected as in A. Seventy-two hours later, total mRNAs were extracted and subjected to RT-qPCR for the indicated genes.

C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.

D U2OS cells were transfected with control or NAT10 siRNAs. Seventy-two hours later, cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 (upper panels). Beta-actin was evaluated as a loading control (lower panel). p53 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.

E U2OS cells were treated with MG132 (10μM) for 4 hours after transfected with the indicated siRNAs. Proteins from cell extracts were detected by western blot for the indicated proteins.

F HCT116 p53+/+ cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibody and immunoblotted with anti-Ub antibody.

G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.

H U2OS cells were transfected with vectors as indicated. Forty-eight hours later, cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies.

Error bars represent the SEM of triplicate experiments.
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 4

Figure 4 NAT10 is a novel E3 ligase for Mdm2

A HCT116 cells were transfected with increasing amount of DNA expressing NAT10. Forty-eight hours later, whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies.

B HCT116 cells were transfected with Flag-NAT10 and harvested at indicated time points after treated with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative Mdm2 protein levels standardized by β-actin are shown in the right. Mdm2 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.

C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours. The Mdm2 protein was immunoprecipitated with anti-Mdm2 antibody and immunoblotted with anti-HA antibody.

D In vitro ubiquitination was performed with recombinant GST-Mdm2 (left) or GST-Mdm2 C464A mutant (right) and Flag-NAT10 or Flag-NAT10 GE in the presence of E1, E2 and Ub in vitro as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE, followed by western blot for evaluation of the indicated proteins.

E Mdm2 is ubiquitinated by NAT10 in a dose-dependent manner. In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag-NAT10 as described in D.

F Schematic representation of NAT10 deletion mutant constructs.

G U2OS cells transfected with NAT10 siRNA-3 (targeting 3’-UTR of NAT10 mRNA) or control siRNA were co-expressed different NAT10 deletion mutants. Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies. Relative p53 protein levels standardized by β-actin are shown in the lower.

H In vitro ubiquitination reaction was conducted using recombinant GST-Mdm2 C464A and full-length Flag-NAT10 or Flag-NAT10-D5. Ubiquitinated Mdm2 was detected as in D.

I HCT116 cells were transfected with the indicated constructs. Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared. Mdm2 protein levels were evaluated by western blot. Relative Mdm2 protein levels standardized by β-actin are shown in the right.

Error bars represent the SEM of triplicate experiments.
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 5

Figure 5 NAT10 regulates p53 activity in DNA damage response

A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies.

B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies.

C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot.

D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins.

E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM.

F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies.

G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM.

H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins.

I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 6

Figure 6 Effects of NAT10 on p53 mediated-transcriptional activity, cell cycle arrest and apoptosis

A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells. Luciferase activity was determined as described in Experimental Procedure.

B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR.

C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student’s t test)

F U2OS cells were transfected with the indicated siRNAs. Cells were irradiated with 50 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Quantification of apoptotic cells is presented. (two-tailed Student’s t test)

G U2OS cells transfected with the indicated siRNAs were plated. Cell numbers were determined as described in Experimental Procedures at the indicated time points.

H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs. The right panel shows the quantification of formed colonies. **p<0.01, ***p<0.001 (two-tailed Student’s t test)

(A-G) Error bars represent the SEM of the triplicate experiments.
NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2 (2016)
Figure 7

Figure 7 NAT10 is downregulated in CRC samples

A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT-qPCR. (N, Normal tissue; T, Tumor tissue) **p<0.01 (non-parametric Mann-Whitney t-test)

B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues. NAT10-specific immuno-complex was detected with DAB as shown as brown color. Quantification of NAT10-positive or NAT10-negative CRC cases is shown in the lower table.

C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top. Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53.

D CRISPR-Cas9 system mediated NAT10 gene editing. The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures. The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins.

E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student’s t test)

F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies.

G HCT116 Ctrl or c2-4 cells were irradiated with 80 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student’s t test)

H Colony formation assay were performed with HCT116 cells (Ctrl, c1-1 and c2-4). The right panel shows the quantification of formed colonies. (two-tailed Student’s t test)

I HCT116 c1-1 and c2-4 cell lines were plated and cell growth curves were determined as described in Fig 6F. Error bars represent the SEM of the triplicate experiments.

J The working model of p53 regulation by NAT10.
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
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(a–d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (a) The cell viability was assayed by trypan blue staining. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student’s t-test). (b) Cell count and viability assay using Muse (Millipore, USA). Data are mean from three independent experiments, with error bars representing s.d. *P0.05 (Student’s t-test). (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student’s t-test). (d) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49. Relative dUTX expression levels following knockdown using the two independent dsRNA are shown (n=3) with error bars representing s.d. *P0.05 (Student’s t-test).
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
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(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX–NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX–NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows. (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies. (c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
f3

(a) Histological analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data was at 24 h RPF. (b,c) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF. (b) Cleaved caspase-3 antibody (green) and Lamin antibody (red) staining are shown in control and dUTX1 salivary glands at 14 h RPF. Scale bar represents 50 μm. (c) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD-AMC as a substrate. Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts. The error bars represent s.e.m. *P-value0.05 (Student’s t-test). (d) Autophagy puncta examined using mCherry-Atg8a (red) and nuclei stained with Hoechst (blue). Scale bar represents 100 μm. The quantitation of the mCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell (control n=242, dUTX1 n=224), with error bars representing s.e.m. *P-value0.05 (Student’s t-test).
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
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(a) Levels of dark, dronc, drice and rpr transcripts were measured from third instar larvae (L3), 6 h and 14 h RPF salivary glands from control (blue) and dUTX1 (red) by qPCR and expressed relative to the internal control gene rp49. (b) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control (blue) and dUTX1 (red) were analysed by qPCR at L3, 6 h and 14 h RPF. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student’s t-test).
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
f5

(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of dark, dronc, drice and rpr (a) and Atg1, Atg2, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg9, Atg12 and Atg18 (b), expressed relative to the internal control gene Gapdh. Results are shown as the fold enrichment of % input compared with IgG control. (c) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP. SL2 cells knocked down for dUTX were chromatin-immunoprecipitated with anti-H3K27me3 and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. (d) The occupancy of EcR on the promoter regions of dark, dronc and Atg1 by ChIP expressed relative to the internal control gene rp49. Chromatin was immunoprecipitated with anti-EcR-B1 and anti-GFP (IgG control) antibodies from SL2 cell following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student’s t-test).
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
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(a) Levels of BR-C and E93 transcripts were measured in salivary glands isolated from third instar larvae (L3), 6 h and 14 h RPF control (blue line) and dUTX1 (red line) using qPCR and expressed relative to the internal control gene rp49. (b) The occupancy of dUTX on the promoter regions of BR-C and E93 genes detected by ChIP. SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. In the right hand panel abundance of H3K27me3 on the promoters of BR-C and E93 genes as detected by ChIP is shown. SL2 cells knockdown for dUTX (dUTX RNAi) were chromatin-immunoprecipitated with anti-H3K27me3 and rabbit IgG (control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Students t-test).
UTX coordinates steroid hormone-mediated autophagy and cell death (2013)
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(a) Mosaic clones of dUTX1 (hsFLP; ubi-GFP FRT40A/dUTX1 FRT40A) from third instar larval salivary glands with wild-type cells marked by nuclear-localized GFP (green). dUTX1 clones have high levels of H3K27me3 (red) compared with the neighbouring control cells marked by GFP. Scale bar represents 50 μm. (b) Western blot analysis of the level of H3K27me3 in control and dUTX1 larval salivary glands. Alpha-tubulin and H3 serve as loading controls. (c) Expression of dUTX wild-type (dUTXWT) and dUTX catalytic mutant (dUTXJmj) in the salivary glands (using Sgs3-GAL4 driver) of dUTX1. Histological analysis of paraffin sections at 24 h RPF shows intact salivary glands present in dUTX1 and in dUTX1; dUTXJmj* compared with control and dUTX1; dUTXWT at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data at 24 h RPF is shown on the right. (d) A schematic representation of the role of dUTX in ecdysone-mediated transcriptional regulation of cell death genes, directly or via E93.
Decreased serum level of sphingosine-1-phosphate: a novel predictor of clinical severity in COVID-19 (2020)
Figure 1

Figure 1. Serum levels of S1P and apoM in HLT and COV patients.

A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.

C-F: Pearson correlation between S1P and apoM (C), RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.
Decreased serum level of sphingosine-1-phosphate: a novel predictor of clinical severity in COVID-19 (2020)
Figure 2

Figure 2. Correlations between S1P/apoM and their transporters

A, B: correlations between S1P and HDL-C (A), and albumin (B). C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.
Decreased serum level of sphingosine-1-phosphate: a novel predictor of clinical severity in COVID-19 (2020)
Figure 3

Figure 3. Serum levels of S1P and its blood transporters in noICU and ICU patients.

(A-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.
Decreased serum level of sphingosine-1-phosphate: a novel predictor of clinical severity in COVID-19 (2020)
Figure 4

Figure 4. Correlation between S1P/apoM and COVID-19 infection severity.

(A-F) Pearson correlation between S1P (A-C) or apoM (D-F) and PSI (A, D), days from admission (B, E) and NLR (C, F) in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.
Using cellular fitness to map the structure and function of a Major Facilitator Superfamily effluxer (2017)
Figure 5

Figure 5. Statistics of ratio γ/κ allow us to test first order approximation assumptions. A. The spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ. B. The calculated mean and error of γ/κ for the sampled parameter fittings shown in Figure S2. The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $\frac{\gamma}{\kappa}\ \approx k_{1}\frac{{2P}_{\text{tot}}}{D_{2}}$.
Using cellular fitness to map the structure and function of a Major Facilitator Superfamily effluxer (2017)
Figure 7

Figure 7. The physicochemical-fitness model can be used to screen plasmid encoded libraries of protein variants. A. Triplicate normalized fitness functions (*) for E. coli BW25113 strains harboring plasmid copies of tet(B)WT, tet(B)K183E,A290V,T326I (variant 6) and tet(B)F151I,F179L,T326A (variant 7) in TET and their corresponding least squares model fits (solid line) where both variants display decreased fitness in TET compared to WT. B. Percent change relative to WT in parameters κ and γ for strains expressing tet(B)K183E,A290V,T326I and tet(B)F151I,F179L,T326A. Parameter values were determined by the least squares model fits in 7A. C. Homology model of TetB using YajR (PDB 3wdo) built using Phyre2 where the periplasmic side is on top and the cytoplasmic side is on the bottom. The A-helices are shown in blue, B-helices are shown in green, C-helices are shown in grey and the interdomain loop is cyan. Spheres indicating variant positions were made using PyMOL (K183 and A290V- red; F151 and F179- red; and T326-black as this residue is altered in variant 6 and 7). The right image depicts the view through central TetB pore from the periplasm into the cytoplasm.