EMBO/soda-nel huggingface dataset:
Modulation of intracellular ROS levels by TIGAR controls autophagy. (2009)
Figure 6

TIGAR modulation of autophagy influences apoptosis. (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).
Blocking interaction between SHP2 and PD-1 denotes a opportunity for developing PD-1 inhibitors (2020)
Figure 1

Fig. 1. MB enhances cytotoxicity of activated CTL against PD-L1 expressing target cells

Structural formula of 3,7-Bis(dimethylamino)-5-phenothiazinium Chloride (MB).

Impact of MB on cytotoxicity of OT-1 CTLs against EG7 or EG7-L1. Splenocytes from OT-1 mice in culture were stimulated with 10 nM of OVA257-264 (SIINFEKL) for 3 days to generate mature CTLs. CTLs were incubated with CFSE labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry analysis. Data are representative of three independent experiments. (effector to target ratio = 5:1, unpaired t-test). EG7-L1: EG7 overexpressing PD-L1.

Statistical results of (B).

ELISA measured of LDH release to assess the cytotoxicity of OT-I CTLs on EG7-L1 in the presence of MB. Anti-PD1 antibody (aPD-1) served as positive control.

Impact of MB on cytotoxicity of PD-1-/- (PD-1KO) OT-1 CTLs against EG7 or PD-L1 expressing EG7 stable cell line. Data are representative of three independent experiments. (unpaired t-test).

Statistical results of (E).

Cytotoxicity of OT-I CTLs against B16-F10-OVA cells (designated B16-OVA) in the presence of 1 μΜ of MB or DMSO. Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope (scale bar = 50 μm). Data are representative of three independent experiments, and were analyzed by unpaired t-test.

Statistical results of (G).

Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Blocking interaction between SHP2 and PD-1 denotes a opportunity for developing PD-1 inhibitors (2020)
Figure 2

Fig. 2. MB enhanced activation and effector function of CTL

Effect of MB on the proliferation of WT CTLs, PD-1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28 coated 96-well plates. 10 μg/ml of PD-L1 were administered in media. Cell proliferation were checked by monitoring dilution of CFSE 48 hours after CD3/CD28 stimulation by gating on CD8 positive population through FACS analysis. WT: splenic cell from wildtype C57BL/J mice. PD-1KO: splenic cell from PD-1 knockout mice.

Statistical results of (A).

FACS analysis of the effect of MB on the activation of OT-I CTLs. OT-I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD-L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 hours, surface expression of CD25 and CD69 on OT-1 CTLs were determined through FACS analysis.

Statistical results of (C).

Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP-luc by co-culture with Raji-L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE-Loaded Raji (PD-L1 negative) served as positive control). IC50 is calculated to be 117 nM. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1.

Impact of MB on luciferase activity of various engineered Jurkat T cells. J-luc: Jurkat cell harboring NFAT-luciferase transgene; J-luc-sgPD-1: J-luc cells treated with lentivirus expressing sgPD-1/CAS9 simultaneously.

qRT-PCR analysis of IL-2 mRNA level in JP-luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD-L1 and MB at indicated concentrations.

MB enhancing IL-2 expression by JP-luc stimulated with Raji-L1 for 24-hour quantified through ELISA analysis (aPD1 served as positive control).

MB enhancing production of cytokine and cytolytic granule by OT-I CTLs. CTLs were incubated with EG7-L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule were determined by flow cytometry. aPD1 served as positive control. EG7-L1: EG7 overexpressing PD-L1.

Statistical results of (I).

Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Blocking interaction between SHP2 and PD-1 denotes a opportunity for developing PD-1 inhibitors (2020)
Figure 3

Fig. 3. MB suppress PD-1 signaling through blocking SHP2 recruitment by PD-1

Confocal cell images of Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry. Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry were incubated with 10 μg/ml of human PD-L1 protein in the presence of 100 nM and 1 μM of MB for 2 minutes, fixed with 4% paraformaldehyde and stained with DAPI. Colocalization signal were examined with confocal microscope (scale bar = 2 µm).

Fluorescence complementation analysis of the effect of MB on the interaction between PD-1 with SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD-L1 protein in the presence of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity.

Co-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 hours.

Ability of MB to block interaction between PD-1 with SHP2 revealed through ELISA. Y248 phosphorylated human PD-1 ITSM peptide was coated at 4 μg/ml in 96 plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 hours. Bound SHP2 were measured by monitoring the activity of horse-peroxidase conjugated on secondary antibody targeting SHP2.

Representative western blot showing the levels of total and phosphorylated ZAP70, PKCθ and PLCγ1 in the lysates of stimulated CTL, inhibited by PD-L1 in the presence of 1μΜ of MB.

Representative western blot showing the levels CD28 phosphorylation. CTL were stimulated with EG7 cells (parental), EG7-L1 and EG7-L1 in the presence 100 nM of MB for 2 hours. Protein immunoprecipitated (IP) with Anti-pY from the lysates of the indicated CTL-EG7 co-culture were subjected to SDS-PAGE separation and blotted with aCD28. β-actin served as loading control. EG7-L1: EG7 overexpressing PD-L1.

Phos-tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7-L1 in the presence of 1μΜ MB. Cell lysate were then separated by SDS-PAGE containing 50 μM of Phos-tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow-moving) species were visualized through exposure.

Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD-L1 protein and 100 nM of MB for 6 hours. Luciferase activity was measured as readout for CD28-P85 interaction.

Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001.

Source data are available online for this figure.
Blocking interaction between SHP2 and PD-1 denotes a opportunity for developing PD-1 inhibitors (2020)
Figure 4

Fig. 4. MB shrinks tumor in vivo through enhancing cytotoxic function of CTL

Schematic of the xenograft mouse model for MB treatment. C57BL/6J mice were inoculated with EG7-L1 cells (2 × 106 cells, s.c.) on the right flank on day 1, followed by injection (2 × 106 cells, i.v.) of CD45.1+ CTL on day 3 and day 6 respectively. The mice were randomized into 3 groups (n = 5) and treated with vehicle, MB (20 mg/kg/day, i.g.), aPD1 (10 mg/kg, every other day, i.p.) served as positive control. Tumor size was measured through calipering every other day. EG7-L1: EG7 overexpressing PD-L1.

Tumor growth curve for (A) was shown as mean ±s.e.m.

Representative image of the tumor on day 10 mentioned in (A).

Tumor weight on day 10 (n=5) mentioned in (A).

Immunohistochemical analysis of cell proliferation marker ki-67 and CD8 in tumor nodules on day 10 (n=5) in (A).

Statistical results of (E).

Expression of Perforin, GZMB and IL-2 by CD8+ CD45.1+ TILs were analyzed by flow cytometry.

Statistical results of (G).

Schematic of EGFR L858R mice treated with MB.

Tumor burdens recorded through Computed tomography (CT) scanning for EGFR L858R mice (n=5, lung section = 3). Mice were treated with MB (20 mg/kg/day, i.g.) for 1 week, followed by imaged with CT again to record tumor burden.

Statistical results of (J).

Haematoxylin and Eosin staining of lung section of EGFR L858R mice (n = 5, lung section = 3) in (J).

Statistical results of (L).

Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Blocking interaction between SHP2 and PD-1 denotes a opportunity for developing PD-1 inhibitors (2020)
Figure 5

Fig. 5. MB is effective to activate human CD8+ T cells

Effect of MB on proliferation of human CD8+ T cells. CFSE labeled human peripheral blood mononuclear cells (PBMC) were pre-incubated with DMSO, MB, 25 μg/ml of pembrolizumab or 20 μg/ml of Nivolumab for 1 h and then seeded in the 96-well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD-L1 Protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE.

Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations.

Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1.

Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.
Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. (2020)
Figure 1.

(a) Phylogenetic analysis of 2019-nCoV spike glycoprotein from its protein BLAST sequences. The neighbour-joining tree was constructed using MEGA X, tested by bootstrap method of 2000 replicates, and edited by the online tool of iTOL (v5). (b) The simulated model of 2019-nCoV RBD binding to SARS-CoV-RBD-specific antibodies (m396, 80R, and F26G19). (c) Protein sequence alignment of 2019-nCoV and SARS-CoV RBD, showing the predominant residues that contribute to interactions with ACE2 or SARS-CoV-specific antibodies. (d) The comparison of the complex structures of SARS-CoV-RBD and SARS-CoV-RBD-specific antibodies (shown in the first row) and models of 2019-nCoV-RBD and SARS-CoV-RBD-specific antibodies (shown in the second row). (e) Binding of monoclonal antibodies to 2019-nCoV RBD determined by ELISA. (f) Binding profiles of 2019-nCoV RBD to ACE2 and antibodies, and (g) competition of CR3022 and ACE2 with 2019-nCoV RBD measured by BLI in OctetRED96. Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.
Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis (2007)
figf2

(a–c) We used quantitative real-time PCR to determine the expression patterns of ATG5 (a), ATG7 (b) and ATG16L1 (c) in various human immune and epithelial cell lines. ATG5 was broadly expressed in the cell lines studied (a), with between 1.5- and fourfold variations in expression level across the cell lines. ATG7 expression (b) was broadly comparable to that of ATG5, with the exception of THP1 cells, which showed 35-fold higher expression than SW480 cells. ATG16L1 expression (c) was also broadly similar to that of ATG5 and ATG7 (except for ATG7 expression in THP1 cells), with moderate variations across the cell lines tested (1.5- to 3.5-fold). (d) Expression of ATG16L1 in human primary immune cells. In a resting human immune cell RNA panel (Clontech), ATG16L1 showed a clear peak of expression in the T cell compartment, with CD4+ and CD8+ cells expressing high levels of transcript (15- to 20-fold more than control samples). We detected modest levels of ATG16L1 in CD19+ cells and low levels in CD14+ monocytes and unfractionated mononuclear cells. In all cases, we performed real-time quantitative RT-PCR reactions in duplicate and plotted the means; error bars represent 1 s.d. We normalized expression levels by comparison with GAPDH controls and plotted arbitrary relative expression units, where the SW480 or placental RNA expression equals 1 (for cell lines or primary cells, respectively). We isolated all RNAs from resting cells in the absence of stimulation or activation.
Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis (2007)
figf3

(a) We achieved specific siRNA knockdown of overexpressed, Flag-tagged ATG16L1 in HEK293 cells within 48 h of transfection with oligonucleotide duplexes. Flag-ATG16L1 was undetectable by protein blotting after treatment with specific siRNA constructs, but expression was maintained with control duplexes. (b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance ± s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments. (c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (± s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances. (d) Representative images of intracellular S. typhimurium show normal autophagic encapsulation by LC3+ membranes in control siRNA–treated cells but loss of autophagic targeting after ATG16L1 knockdown. Channels are separated to show both the LC3-GFP fusion protein (autophagosomal marker) and S. typhimurium SL1344 dsRed2 (SL1344), with a merged image in the final column. Rows 2 and 4 show magnified views of boxed areas in rows 1 and 3, respectively. Images were obtained using confocal microscopy and are a flat projection of z stacks taken throughout the plane of the infected cell. Scale bars = 5 μm.
Protein abundance of AKT and ERK pathway components governs cell-type-specific regulation of proliferation (2017)
Figure 2

Figure 2. Mathematical modeling of the Epo-induced AKT, ERK and S6 activation.

A The model is represented as a process diagram and reactions are modulated by enzyme catalysis (circle-headed lines) or inhibition (bar-headed lines). Prefix ‘p’ represents phosphorylated species. Bold framed species were experimentally measured. White capsules represent inhibitors. Binding of the ligand Epo to its cognate receptor results in the phosphorylation of EpoR. Receptor and associated complexes are depicted in yellow, the AKT pathway is red, the ERK pathway is blue, and cell cycle genes and the S6 network is shown in green.

B-G Model calibration with time-resolved quantitative immunoblot data of mCFU-E cells in blue and BaF3-EpoR cells in black. Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR (B), pAKT (C), ppERK (D). The scale for pS6 (E) was estimated in arbitrary units. GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting. For pAkt and ppERK, cellular lysates were subjected to quantitative immunoblotting. Calibrator proteins were used for EpoR, AKT, GTP-Ras and ERK to facilitate the conversion to nM concentrations. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.
Protein abundance of AKT and ERK pathway components governs cell-type-specific regulation of proliferation (2017)
Figure 3

Figure 3. Model predictions and experimental validations for negative regulators of AKT and ERK signaling.

A Model calibration with mCFU-E wild-type cells, and mCFU-E cells overexpressing SHIP1 or PTEN, and BaF3-EpoR wild-type cells. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.

B Model prediction of pAKT dynamics in BaF3-EpoR cells overexpressing PTEN or SHIP1. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.

C Model prediction for the basal expression level of the dual specific phosphatase (DUSP). DUSP abundance ratio between BaF3-EpoR and mCFU-E cells was identified by the mathematical model. The solid line indicates the profile likelihood. The dashed red line indicates the threshold to assess point-wise 95% confidence interval. The asterisk indicates the optimal parameter value.

D Experimental validation of basal DUSP expression in mCFU-E and BaF3-EpoR. Quantitative RT-PCR was performed with all samples on the same plate, allowing a direct comparison of mRNA levels between mCFU-E and BaF3-EpoR cells. Data is normalized to the Rpl32 gene. Ratios of expression in BaF3-EpoR cells compared to mCFU-E cells are shown as box plots. N=10. For details, see Appendix K.
Protein abundance of AKT and ERK pathway components governs cell-type-specific regulation of proliferation (2017)
Figure 4

Figure 4. Validation of Epo-induced signaling dynamics in 32D-EpoR cells under RSK wild-type conditions and upon overexpression of RSK.

A Model prediction and experimental validation for pAKT, ppERK and pS6 dynamics of 32D-EpoR cells in response to 50 U/ml Epo. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.

B Sensitivity analysis for integrated pS6 in mCFU-E, 32D-EpoR and BaF3-EpoR cells. Measured, cell-type-specific protein abundance and estimated global kinetic rates were taken into account. Proteins were grouped according to the network modules. Integrated pS6 exhibited high sensitivity towards RSK in 32D-EpoR and BaF3-EpoR cells but not in mCFU-E cells.

C Model prediction and experimental validation for RSK, AKT, ERK and S6 activation upon RSK overexpression (oe) and 5 U/ml Epo stimulation in 32DEpoR cells. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D-EpoR wild-type (wt) cells, N=4 (lower right panel). Two-Sample t-Test, *** p<0.005.

D Impact of AKT, Ras or PTEN overexpression on Epo-induced pS6 dynamics in mCFU-E and BaF3-EpoR cells. Quantitative immunoblotting upon overexpression of PTEN, AKT, a constitutive active Ras protein, or the empty vector control in mCFU-E cells or BaF3-EpoR cells. Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with 5 U/ml Epo for indicated time points. Cellular lysates were analyzed by immunoblotting employing sequential reprobing anti-pAKT, anti-ppERK, anti-pS6, anti-S6 and to ensure equal loading with anti-beta-actin antibodies. Detection was performed with chemiluminescence using a CCD camera device (ImageQuant). Quantification of pS6 on the right is depicted as fold change to wild-type samples at 30 minutes after Epo stimulation. Error bars represent standard deviation. oe: overexpression. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05.