Journal: Advanced Science

Article Title: VDLIN: A Deep Learning‐Based Platform for Methylcobalamin‐Inspired Immunomodulatory Compound Screening

doi: 10.1002/advs.202413775

Figure Lengend Snippet: Co7 induces Ifnb1 expression via the TLR4 signaling pathway. A) Volcano plot illustrating the distribution of differentially expressed genes (DEGs) between the Co7 and DMSO treatment groups after 3 h in RAW 264.7 cells. Fold changes are presented as log 2 transformations. Red dots represent DEGs upregulated in the Co7 group. B) Protein‐protein interaction (PPI) network analysis of differentially expressed genes (DEGs) in the Co7‐treated group compared to the DMSO group, revealing significant enrichment in pathways associated with the innate immune response and type I interferon signaling. C) KEGG pathway enrichment analysis of DEGs induced by Co7, highlighting associations with innate immune response pathways. D) Co7 (50 µmol/L) exhibiting strong antiviral effects against VSV in RAW 264.7 and HT29 cells. E) Co7 significantly reduced the inflammatory response induced by LPS, VSV, EMCV, and HSV in RAW 264.7 cells. F) Volcano plot representing the differential gene expression analysis between Co7‐ and LPS‐treated RAW 264.7 cells after 3 h of treatment. Fold changes are displayed as log 2 transformations. Red dots indicate genes upregulated in the Co7 group, while blue dots represent downregulated genes compared to LPS treatment. G) Western blot analysis demonstrating that Co7 inhibited the expression of iNOS and COX2, as well as the phosphorylation of NF‐κB‐P65 at the protein level in RAW 264.7 cells. H) Co7 significantly reduced the mortality rate in mice (n = 10 per group) following LPS challenge (20 mg/kg), compared to the PBS control group. RT‐qPCR data were presented as means ± SEM from three independent experiments. Statistical significance was determined using one‐way ANOVA with Bonferroni's multiple comparisons test (left three panels in E), paired‐samples t‐test (D, right panel of EMCV and HSV in E), or the log‐rank test (H). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Each inhibitor was dissolved in anhydrous DMSO and diluted to its respective working concentration: C29 (10 μM, S6597, Selleck) served as a TLR2 inhibitor; Procyanidin B1 (30 μM, HY‐N0795, MedChemExpress) acted as a TLR4 inhibitor; MyD88‐IN‐1 (30 μM, HY‐149992, MedChemExpress) was used to inhibit MyD88; Pepinh‐TRIF TFA (30 μM, HY‐P2565, MedChemExpress) functioned as a TRIF inhibitor; and GSK8612 (5 μM, T5540, TargetMol) inhibited TBK1/IKKε.

Techniques: Expressing, Gene Expression, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR

Journal: Blood

Article Title: MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

doi: 10.1182/blood.2019003940

Figure Lengend Snippet: STAT3 transactivates Mdh1 expression to maintain FL-HSC activities. (A) Mdh1 luciferase reporter and different doses of Stat3 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (B) ChIP assays were analyzed with 293T cells cotransfected with pGL4.27-mdh1 promoter vector and Stat3 plasmid or control plasmid. Input control and amplification of the Stat3-binding sequence of Mdh1 were determined by semiquantitative PCR. (C) Bisulfite sequencing analysis of the methylation patterns of the CpG islands in the Mdh1 promoter in CD45+ SoNar-high and -low FL hematopoietic cell fractions is shown. Each row represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. (D) Quantification data of methylated and unmethylated CpGs (n = 3). (E) Protein levels of pSTAT3, STAT3, and MDH1 were measured in CD45+ SoNar-high and -low FL cells. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against SoNar-high#1 cells. (F) Representative flow cytometric analysis of intracellular level of pSTAT3 of total CD45+ FL hematopoietic cells and FL-HSCs. Mean fluorescence intensity (MFI) of each group was shown (isotype control, yellow and green lines). (G) Quantification of MFI of intracellular level of pSTAT3 in panel F (n = 3). (H) Immunoblotting assay showed protein levels of pSTAT3, STAT3, and MDH1 in total CD45+ FL hematopoietic cells and FL-HSCs. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against total cells. (I) Representative flow cytometric analysis of intracellular level of pSTAT3 of CD45+ SoNar-high and -low FL-HSCs. MFI of each group is shown (isotype control, yellow and green lines). (J) Quantification of MFI of intracellular level of pSTAT3 in panel I (n = 3). (K) Protein level of MDH1 was measured in Stat3-overexpressed SoNar 32D cells and control cells by immunoblotting. Ratios of STAT3/actin and MDH1/actin were quantified and normalized against control cells. (L) Stat3-overexpressed SoNar 32D cells and control cells were evaluated for the ratios of SoNar fluorescence, and representative images are shown. Scale bar, 10 μm. (M) Quantification of the ratios of SoNar fluorescence in panel L. A total of 30 SoNar 32D cells were analyzed (n = 3). (N) Working model for the connections between FL-HSC activities and Mdh1-mediated malate-aspartate shuttle as indicated by SoNar. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G,J,M) and 2-way analysis of variance with Sidak’s multiple comparison test (D) were used for the comparison. *P < .05, ***P < .001.

Article Snippet: To test the effect of STAT3 inhibitor on MDH1 expression, purified Lin − Sca-1 + c-Kit + FL-HSCs and adult HSCs were treated with 10 or 20 μM of STAT3 inhibitor C188-9 (TargetMol) for 24 hours according to the previous study 37 and subjected to determination of pSTAT3, STAT3, or MDH1 level by western blot.

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Amplification, Binding Assay, Sequencing, Methylation Sequencing, Methylation, Fluorescence, Western Blot, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: ASAP and Plk1 co-localize at centrosomes and interact in vivo during mitosis. A and B, asynchronous U-2 OS cells were grown on glass coverslips and fixed in PAF/MTSB (A) or F/PHEM/methanol (B) (see “Materials and Methods”) and labeled with the polyclonal anti-ASAP (green), or the monoclonal anti-Plk1 antibodies (red) and stained with Hoechst 33258 (blue) without (A) or after (B) depolymerization of MTs by incubation on ice for 60 min. The different stages of the cell cycle are shown from prophase (A) or interphase (B) to telophase (scale bar, 10 μm). C, U-2 OS cell lysates were immunoprecipitated (IP) with rabbit IgG, rabbit anti-ASAP, or anti-Myc antibodies, and the precipitates were analyzed by immunoblotting (IB) with antibodies against ASAP or Plk1. Left, asynchronous cells were co-transfected with FLAG-ASAP and Myc-Plk1 (Input, 10% of protein extracts; immunoprecipitated with 200 μg of protein extracts). Right, endogenous proteins were co-immunoprecipitated from synchronized mitotic cells (see “Materials and Methods”) (Input, 10% of protein extracts; immunoprecipitated with 2 mg of protein extracts). A higher exposure is shown for Plk1 immunoblot. See supplemental Fig. S1A for uncropped gels. D, asynchronous U-2 OS cells were transfected with FLAG-ASAP and either Myc-Plk1-WT (left) or Myc-Plk1-PBD mutant (mut) (right) and immunoprecipitated with rabbit IgG or rabbit anti-ASAP antibody. Precipitates were analyzed by immunoblotting with antibodies against ASAP or Plk1. See supplemental Fig. S1B for uncropped gel. E, U-2 OS cells were transfected with FLAG-ASAP, and 24 h later, cell lysates were immunoprecipitated with rabbit IgG or rabbit anti-ASAP antibodies (Input, 10% of protein extracts immunoprecipitated with 200 μg of protein extracts). Precipitates were analyzed by immunoblotting with antibodies against ASAP, Plk1, or Aurora A. See supplemental Fig. S1C for uncropped gel. F, FLAG-ASAP-transfected cells were arrested at the G1/S boundary by thymidine block (2 mm for 24 h) and then released into fresh medium. Samples harvested at the indicated time points were analyzed by immunoblotting using antibodies against ASAP, Plk1, or Aurora A. Inputs are shown on the left and immunoprecipitates on the right. α-Tubulin was used as a loading control.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: In Vivo, Labeling, Staining, Incubation, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Blocking Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Plk1 promotes ASAP localization to centrosomes and spindles through the NEDD1-γ-tubulin pathway. A, asynchronous U-2 OS cells were transfected with Luciferase GL2 (control), ASAP, or Plk1 siRNAs. Cell extracts were immunoblotted (IB) and probed with anti-ASAP, Plk1, and α-actin (loading control) antibodies. For immunofluorescence analysis, asynchronous U-2 OS cells grown on coverslips were transfected with GL2 or Plk1 siRNAs or incubated with 1 μm TAL overnight. Cells were fixed in PAF/MTSB (left panel) or F/PHEM/methanol (right panel), probed with the polyclonal anti-ASAP (green) and the monoclonal anti-Plk1 antibody (red), and stained with Hoechst 33258 (blue) without (left panel) or after (right panel) MT depolymerization by incubation on ice for 60 min (scale bar, 10 μm). The percentage of mitosis with aberrant localization of ASAP is shown in the table. B, asynchronous U-2 OS cells were transfected with GL2 or γ-tubulin siRNAs. Cell extracts were immunoblotted and probed with the anti-ASAP, anti-γ-tubulin, and α-actin (loading control) antibodies. For immunofluorescence analysis, cells were fixed in F/PHEM/methanol, probed with the polyclonal anti-ASAP (green) and the monoclonal anti-γ-tubulin antibody (red), and stained with Hoechst 33258 (blue) without (left panel) or after (right panel) MT depolymerization by incubation on ice for 60 min (scale bar, 10 μm). The percentage of mitosis with aberrant localization of ASAP is shown in the table. C, synchronized mitotic cells were immunoprecipitated (IP) with rabbit IgG or rabbit anti-ASAP antiserum, and precipitates were analyzed by immunoblotting with antibodies against ASAP and γ-tubulin (Input, 10% of protein extracts immunoprecipitated with 2 mg of protein extracts). D, centrosomes were isolated and enriched from U-2 OS cells using a discontinuous sucrose gradient (see “Materials and Methods”). Left panel, anti-ASAP and γ-tubulin antibodies were used for Western blot analysis. Right panel, centrosomes from fraction 2 were spun down onto coverslips and analyzed by immunofluorescence using the polyclonal anti-ASAP (green) and the monoclonal anti-γ-tubulin antibodies (red).

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Transfection, Luciferase, Control, Immunofluorescence, Incubation, Staining, Immunoprecipitation, Western Blot, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Plk1 phosphorylates ASAP on S289 in vitro. A, purified, recombinant GST-ASAP, α-casein, or GST was incubated with purified His6-tagged wild-type Plk1 (His6-Plk1-WT) or the kinase-dead mutant (N281A) (His6-Plk1-KD) and [γ-32P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography (upper panel); the gel was also stained with Coomassie Blue (lower panel). B, purified GST, GST-ASAP fragments G1–G6 or GST-ASAP were used in a kinase assay with His6-Plk1-WT as described in A and analyzed by SDS-PAGE and autoradiography (upper panel); the Coomassie Blue-stained gel is shown in the lower panel. C, in vitro kinase assays were performed using purified GST-ASAP, GST-ASAP mutants (S289A or S369A/S370A), or GST as described in A and analyzed by SDS-PAGE and autoradiography (upper panel); the Coomassie Blue-stained gel is shown in the lower panel.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: In Vitro, Purification, Recombinant, Incubation, Mutagenesis, SDS Page, Autoradiography, Staining, Kinase Assay

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Phosphorylation of ASAP at Ser-289 by Plk1 in vivo occurs at centrosomes during mitosis. A, U-2 OS cell lysates transfected with FLAG-ASAP-WT or FLAG-ASAP-S289A were immunoblotted (IB) with the anti-ASAP-P-S289 (left) and anti-ASAP (right) antibodies. NT, nontransfected cells. α-Tubulin is shown as a loading control. B, asynchronous U-2 OS cells were grown on glass coverslips, fixed in F/PHEM/methanol, probed with the anti-ASAP-Ser(P)-289 (green) and anti-γ-tubulin (red) antibodies, and stained with Hoechst 33258 (blue); the different stages of the cell cycle are shown from G2 to telophase (scale bar, 10 μm). C, U-2 OS cells were synchronized by double thymidine block and transfected with FLAG-ASAP during the first release, as indicated on the schematic. At the indicated time points after the second release, cells lysates were analyzed by immunoblotting using the indicated antibodies. α-Actin was used as a loading control. D, U-2 OS cells were transfected with FLAG-ASAP-WT, synchronized by thymidine block, and released in the presence of either dimethyl sulfoxide (DMSO) (control) or 1 μm TAL for 13 h. Cell extracts were immunoblotted with the anti-ASAP-Ser(P)-289, anti-FLAG, anti-Plk1, and anti-H3-Ser(P)-10 antibodies. α-Actin is shown as a loading control. E, U-2 OS cells were grown on glass coverslips, transfected with YFP-ASAP-WT (left panels) or YFP-ASAP-S289A mutant (right panels), fixed in PAF/MTSB (upper panels) or in F/PHEM/methanol (lower panels), and probed with anti-ASAP (green) and anti-α-tubulin (red) (top), anti-γ-tubulin (red) (bottom) and stained with Hoechst 33258 (blue) (scale bar, 10 μm).

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Phospho-proteomics, In Vivo, Transfection, Control, Staining, Blocking Assay, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Plk1-dependent phosphorylation of ASAP on serine 289 does not require a priming phosphorylation by Cdk1/Cyclin B in vivo. A, purified GST-ASAP, histone H1 (positive control), or GST-SIP (negative control) was incubated with purified Cdk1/Cyclin B complex and [γ-32P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography (upper panel); the gel was also stained with Coomassie Blue (lower panel). B, purified GST-ASAP-WT or GST-ASAP-S289A mutant (negative control) was first incubated or not with Cdk1/Cyclin B complex for 30 min, treated with the CDK1 inhibitor RO-3306 for 10 min, and finally incubated with Plk1 for 30 min (see “Materials and Methods”). Reaction products were analyzed by SDS-PAGE and immunoblotted (IB) with anti-ASAP-Ser(P)-289, anti-ASAP, anti-Cyclin B, and anti-Plk1 antibodies as indicated. C, U-2 OS cells were transfected with FLAG-ASAP-WT, synchronized by thymidine block, and released for 16 h in the presence of nocodazole. Then, mitotic cells were recovered by mitotic shake-off and treated for 30 min with dimethyl sulfoxide (DMSO) (control), 50 μm roscovitine (CDK inhibitor), or 2 μm RO-3306 (CDK1 inhibitor). Cell extracts were immunoblotted with the anti-ASAP-Ser(P)-289, anti-FLAG, anti-Plk1, anti H3-Ser(P)-10 (mitotic marker) and anti-Cdc27 antibodies. The phosphoshift band of anti-Cdc27 is shown as a marker of Cdk activity. α-Actin was used as a loading control.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Phospho-proteomics, In Vivo, Purification, Positive Control, Negative Control, Incubation, SDS Page, Autoradiography, Staining, Mutagenesis, Transfection, Blocking Assay, Control, Marker, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

doi: 10.1074/jbc.M110.144220

Figure Lengend Snippet: Model for the role of Plk1-dependent ASAP phosphorylation in maintaining centrosome integrity. Plk1 is a master regulator of centrosome maturation through phosphorylation and targeting of different PCM components. These various phosphorylations and interactions promote the recruitment of NEDD1, γ-tubulin, and ASAP to the centrosome. Plk1 binds to ASAP via its PBD (not shown) and phosphorylates ASAP on Ser-289. In the absence of Plk1-driven Ser-289 phosphorylation of ASAP, numeral and structural centrosome abnormalities occur, leading to abnormal mitotic spindle formation.

Article Snippet: The Plk1 inhibitor ZK-thiazolidinone (TAL) was described in Ref. 30 and kindly provided by Bayer Schering Pharma AG.

Techniques: Phospho-proteomics