Journal: The EMBO Journal

Article Title: Termination of STING responses is mediated via ESCRT ‐dependent degradation

doi: 10.15252/embj.2022112712

Figure Lengend Snippet: A Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 4.5 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. B Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg/ml DMXAA for 1, 2 or 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. C Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. D Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/ml DMXAA for 1, 2 or 4 h. Cells were fixed and stained for p‐STING (white) and the nucleus (DAPI; blue), before Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as a maximum intensity projection (MIP) of Z stack images. Scale bar = 5 μm. Data shown are representative of three independent experiments. E, F The size (i.e. mean radius) (E) and mean intensity (F) of individual p‐STING punctate regions was quantified using CellProfiler. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where * P < 0.05, **** P < 0.0001. A representative experiment used for quantification is shown in (D). G–I Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/ml DMXAA or 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cells were lysed for RNA purification and the expression of Ifnb1 (G), Isg15 (H) and Il6 (I) was analysed by qPCR. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Sting −/− iBMDMs expressing either WT hSTING or hSTING‐N154S were left untreated or treated with 1 μg/ml doxycycline (Dox) for 2 h. Cells were then washed and left to recover for a further 8 h in the absence or presence of 2 μM TAK243. Cells were lysed for immunoblot with indicated antibodies. Data shown are representative of three independent experiments. Source data are available online for this figure.

Article Snippet: DMSO (Sigma‐Aldrich #D2650), DMXAA (SelleckChem #S1537), 2′3′‐cGAM(PS)2 (InvivoGen #tlrl‐nacga2srs), LPS‐B5 Ultrapure (InvivoGen #tlrl‐pb5lps), Pam3CSK4 (InvivoGen #tltl‐pms), TAK243 (SelleckChem #S8341), H‐151 (InvivoGen #inh‐h151), doxycycline (Sigma‐Aldrich #D9891).

Techniques: Control, Western Blot, Staining, Microscopy, Purification, 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 , Gene set variation analysis of Cavalli’s patient samples (n=763) revealed that subtypes Group3β and 3γ were enriched with GO terms related to mRNA translation and ribosome biogenesis. b, Multiplex IHC on G3-MB patient samples using CDK8, p-4EBP1, c-MYC, RPS12, p-S6, and p-AKT antibodies. p<0.05 in all biomarker groups. Statistical analysis: unparied t-test. c, Representative bioluminescence images of mice treated with TAK-228 (1mg/kg, daily, oral gavage) compared with those of the control cohort. d, Kaplan–Meier survival curves for animals treated with control or TAK-228. Statistical analysis: Log-rank test. e, IHC analyses of cleaved caspase 3 protein expression in xenografts mice. Three mice from each group were sacrificed 18 days after tumor implantation. Original magnification, ×40. f, GSEA plots of representative gene sets involved in mTOR signaling following CDK8 depletion. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. g, Immunoblot showing the levels of p-4EBP1 and p-S6 following CDK8 knockdown. h, Immunoblot showing IC50 RVU120 treatment inhibited the levels of p-4EBP1 across three MB cell lines. i, Immunoblot showing the level of p-S6 upon treatment with various dose of RVU120.

Article Snippet: The CDK8 inhibitors Torin1 and TAK-228 were purchased from MedChemExpress, and RVU120 for animal studies was provided by Ryvu Therapeutics.

Techniques: Multiplex Assay, Biomarker Discovery, Control, Expressing, Tumor Implantation, Western Blot, Knockdown

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: The CDK8 inhibitors Torin1 and TAK-228 were purchased from MedChemExpress, and RVU120 for animal studies was provided by Ryvu Therapeutics.

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