phosphor stat1 ser727  (Cell Signaling Technology Inc)

Journal: Molecular neurobiology

Article Title: CircKat6b Mediates the Antidepressant Effect of Esketamine by Regulating Astrocyte Function.

doi: 10.1007/s12035-024-04420-0

Figure Lengend Snippet: Fig. 6 CircKat6b regulates astrocyte function via stat1. A Protein expression of stat1 and p-stat1 protein in the hip- pocampus in each group. N = 6, vs control group, **P < 0.01; vs CUMS group, #P < 0.05 using one-way ANOVA followed by Tukey’s test. B Effect of circK- at6b overexpression on stat1 and p-stat1 protein expression in the hippocampus in each group. N = 6, vs control-circCon group, **P < 0.01; vs control-circKat6b group, $$P < 0.01; vs CUMS- circKat6b group, ##P < 0.01; vs CUMS + Es-circCon group, &&P < 0.01 using two-way ANOVA followed by Tukey’s test. C Representative double- staining immunofluorescence of p-stat1 and GFAP in brain sections from different groups following esketamine treatment. Scale bar = 20 µm

Article Snippet: The primary antibodies used were GFAP (Proteintech, 1:10000), GABAB1 (Abcam, 1:100), Stat1 (Proteintech, 1:2000), Phosphor-stat1 (Ser727) (Cell Signaling Technology, 1:1000), and β-actin (Proteintech, 1:5000).

Techniques: Expressing, Control, Over Expression, Double Immunofluorescence Staining

Journal: Molecular neurobiology

Article Title: CircKat6b Mediates the Antidepressant Effect of Esketamine by Regulating Astrocyte Function.

doi: 10.1007/s12035-024-04420-0

Figure Lengend Snippet: Fig. 7 A schematic diagram depicting how esketamine affects the function of astrocytes through the regulation of stat1 expression via circKat6b to exert an antidepressant effect

Article Snippet: The primary antibodies used were GFAP (Proteintech, 1:10000), GABAB1 (Abcam, 1:100), Stat1 (Proteintech, 1:2000), Phosphor-stat1 (Ser727) (Cell Signaling Technology, 1:1000), and β-actin (Proteintech, 1:5000).

Techniques: Expressing

Journal: bioRxiv

Article Title: Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma

doi: 10.1101/2024.03.08.584103

Figure Lengend Snippet: a , Half-maximal inhibitory concentration (IC50) determination of various CDK8 inhibitors in D425, D458, and HDMB03 MB cell lines. Unit: μmol. b, IC50 of RVU120 at 72 h in various MB cell lines and normal human astrocytes (NHA) cells. c, Dose-dependent proliferation curve of RVU120 treated primary cultured medulloblastoma cells from a G3-MB patient. d, Immunofluorescence of CDK8 (green) and DAPI (blue) at 40X magnification. D425, D458, and D341 cells were treated with IC50 RVU120 for 48 h. Scale bar, 10 μm. Statistical analysis: Mann-Whitney Wilcoxon test. e, Immunoblot demonstrates time-course analysis of p-STAT1(S727) protein levels with treatment of IC50 RVU120 across D341, D458, D425 MB cell lines. f, Methylcellulose assay in D425 and D458 cells treated with DMSO, IC25, IC50, and IC75 for 2 weeks. Statistical analysis: two-way ANOVA. g, Representative images of neurosphere size in IC40 RVU120 treated D425 or D458 MB cell lines at 1, 5, and 10 days are shown. n = 5. Mean ± SD. Scale bar, 400 μm. Statistical analysis: one-way ANOVA. h, Annexin V apoptosis assay. D425 and D458 cells were treated with IC50 RVU120 for 48 h, stained with Annexin V, and measured by flow cytometry. n = 3. Statistical analysis: student t-test. i, The concentration of the compound in the CSF corresponds to the free concentration in the brain. A ratio of less than one in rodents indicates the possibility of intercellular trapping. Kpuu: Unbound partition coefficient. j, Representative bioluminescence images of mice treated with RVU120 (40mg/kg, daily, oral gavage) compared with those of the control cohort. k, Kaplan–Meier survival curves for animals treated with Control or RVU120. Statistical analysis: log-rank (Mantel-Cox) test.

Article Snippet: Antibodies used for western blot analysis were from the following sources: β-actin (Cell Signaling, 8457, 1:2000), CDK8 (Cell Signaling, 4101, 1:1000), 4EBP1 (Cell Signaling, 9644S, 1:1000), Phosphor-4EBP1 (Cell Signaling, 2855S, 1:1000), STAT1 (Cell Signaling, 9176S, 1:1000), Phosphor-STAT1 (Cell Signaling, 8826S, 1:1000), S6 (Cell Signaling, 2217T, 1:1000), Phosphor-S6 (Cell Signaling, 4858T, 1:1000), Pol II (Cell Signaling, 2629S, 1:1000), and Phosphor-Pol II-Ser2 (Cell Signaling, 13499, 1:1000).

Techniques: Concentration Assay, Cell Culture, Immunofluorescence, MANN-WHITNEY, Western Blot, Methylcellulose Assay, Apoptosis Assay, Staining, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma

doi: 10.1101/2024.03.08.584103

Figure Lengend Snippet: a , Immunofluorescence of CDK8 (green) and DAPI (blue) at 40X magnification. D425 and D458 cells were treated with IC50 RVU120 for 48 h. Mean ± SD. Scale bar, 10 μm. Statistical analysis: Mann-Whitney Wilcoxon test. b, Immunoblot demonstrating the protein levels of p-STAT1 (S727) and total STAT1 in D425 and D458 cells treated with various dose of RVU120. c, Representative images of neurosphere size in BI1347 treated D425 or D458 MB cell lines at 10 days are shown. n = 5. Mean ± SD. Scale bar, 400 μm. Statistical analysis: One-way ANOVA. d, Flow cytometric analysis of apoptotic population measured by active caspase 3. D425 and D458 cells were treated with IC50 RVU120 IC50 for 48 hours. Mean ± SD. Statistical analysis: One-way ANOVA. e , IHC analysis of cleaved-caspase3 in D458 xenograft mice treated with RVU120 compared to those treated with vehicle. Three mice in each group were treated for 10 days after tumor implantation. Original magnification, ×40.

Article Snippet: Antibodies used for western blot analysis were from the following sources: β-actin (Cell Signaling, 8457, 1:2000), CDK8 (Cell Signaling, 4101, 1:1000), 4EBP1 (Cell Signaling, 9644S, 1:1000), Phosphor-4EBP1 (Cell Signaling, 2855S, 1:1000), STAT1 (Cell Signaling, 9176S, 1:1000), Phosphor-STAT1 (Cell Signaling, 8826S, 1:1000), S6 (Cell Signaling, 2217T, 1:1000), Phosphor-S6 (Cell Signaling, 4858T, 1:1000), Pol II (Cell Signaling, 2629S, 1:1000), and Phosphor-Pol II-Ser2 (Cell Signaling, 13499, 1:1000).

Techniques: Immunofluorescence, MANN-WHITNEY, Western Blot, Tumor Implantation

Journal: bioRxiv

Article Title: Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma

doi: 10.1101/2024.03.08.584103

Figure Lengend Snippet: a Dose-dependent assay of the combined treatment with RVU120 and Torin1 on Day 5 in D425 and D458 cells. b, Real-time proliferation assay quantifying the combined treatment with RVU120 and Torin1 using the Incucyte Live-Cell Analysis System. c, Heatmap representation of the Fraction Affected (FA) and the Bliss interaction index across the five-point dose range of RVU120 and Torin1 in D425 and D458 cells. Mean values of triple biological experiments are shown. d, The combination index of RVU120 and Torin1 using chou-talalay method. The mean combination index (horizontal bar) was determined from three independent experiments using D425 and D458 cells. The results for each experiment are indicated by symbols. e, Apoptosis assay following combined treatment with RVU120 and Torin1. D425 and D458 cells were treated for 48 h before staining with PI and Annexin V. f, Effects of the combination of RVU120 and Torin1 on protein synthesis markers, phospho-Pol2 and phospho-STAT1, in D425 and D458 cells after 48 h of treatment. g, The nude mice injected with D458 cells were treated with vehicle, RVU120 (40 mg/kg), TAK-228 (1 mg/kg), or their combination. Representative bioluminescence images are shown. Color scales indicate bioluminescence radiance in photons/sec/cm2/steradian. h, Kaplan-Meier survival curve of D458 xenograft mice. The treatment period is indicated by shaded boxes. Statistical analysis: log-rank (Mantel-Cox) test. i, Representative Sagittal T2-weighted turboRARE MRI of D458 xenografted mice at 22 days. White arrows indicate tumors. MRI volumetric analysis (right) demonstrates trend toward decreased tumor volume in each cohort.

Article Snippet: Antibodies used for western blot analysis were from the following sources: β-actin (Cell Signaling, 8457, 1:2000), CDK8 (Cell Signaling, 4101, 1:1000), 4EBP1 (Cell Signaling, 9644S, 1:1000), Phosphor-4EBP1 (Cell Signaling, 2855S, 1:1000), STAT1 (Cell Signaling, 9176S, 1:1000), Phosphor-STAT1 (Cell Signaling, 8826S, 1:1000), S6 (Cell Signaling, 2217T, 1:1000), Phosphor-S6 (Cell Signaling, 4858T, 1:1000), Pol II (Cell Signaling, 2629S, 1:1000), and Phosphor-Pol II-Ser2 (Cell Signaling, 13499, 1:1000).

Techniques: Proliferation Assay, Cell Analysis, Apoptosis Assay, Staining, Injection