Journal: Nature Communications

Article Title: CD44 connects autophagy decline and ageing in the vascular endothelium

doi: 10.1038/s41467-023-41346-y

Figure Lengend Snippet: a Fluorescence analysis of the colocalization of CD44ICD with endogenous STAT3 and core proteins of the PtdIns3K complexes in HUVECs transduced with Ev or CD44ICD. Nuclei were stained with DAPI. Bar = 10 μm. The line scanned profiles at the right of confocal images show the distribution of fluorescence for each channel in the white line in the corresponding confocal images. b Reactivity of CD44ICD or STAT3 with PtdIns3K complex proteins, and STAT3 with CD44ICD in a yeast two-hybrid system. Yeast strain AH109 was co-transformed with a bait plasmid, BD-CD44ICD or BD-STAT3, and a prey plasmid, pGADT7-PtdIns3K complex proteins (AD-ATG14L, AD-BECN1, AD-UVRAG, AD-PIK3C3, and AD-PIK3R4) or pGADT7-CD44ICD, which encodes PtdIns3K complex proteins or CD44ICD fused to the Gal4 activation domain. Co-transformation of BD-Lam/AD-T and BD-P53/AD-T was used as negative and positive controls, respectively. c BiFC analysis of CD44ICD-STAT3 and STAT3-PIK3R4 interactions. HUVECs were transfected with indicated combinations of constructs. Co-transfection of VN-Jun and VC-Fos was used as positive control. Bar = 30 μm. d , e GST-pull-down of recombinant STAT3 with a GST-CD44ICD fusion protein ( d ) or a GST-P150 domain fusion protein ( e ). f The binding mode of full-sequence structures of STAT3 and CD44ICD. Colors indicate: STAT3, green; CD44ICD, red. The key residues interacting between STAT3 and CD44ICD are indicated as gray and yellow, respectively. Hydrogen bond is described by pink dash lines. g Western blots showing co-IP of CD44ICD_ΔN35 with endogenous STAT3 and core proteins of the PtdIns3K complexes in HUVECs transduced with Ev or CD44ICD_ΔN35 (DDK tag). Lysates, whole cell lysates; IP, immunoprecipitates. h Proposed schematic diagram of CD44ICD-mediated autophagy decline. CD44ICD suppresses the levels of PIK3C3 and PIK3R4 and the kinase activity of PIK3C3, activates STAT3, and disrupts the assembly of the PtdIns3K complex by interacting with STAT3. Three biologically independent experiments. Source data are provided as a Source data file.

Article Snippet: For the BiFC assay, the sequences of CD44ICD, PIK3R4 and STAT3 were cloned and inserted into both VN155 and VC155 (Addgene, MA, USA).

Techniques: Fluorescence, Transduction, Staining, Transformation Assay, Plasmid Preparation, Activation Assay, Transfection, Construct, Cotransfection, Positive Control, Recombinant, Binding Assay, Sequencing, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: Nature cell biology

Article Title: Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression.

doi: 10.1038/s41556-021-00820-9

Figure Lengend Snippet: Fig. 3 | Tumour-cell-derived GABA induces β-catenin signalling to support proliferation. a, Growth curves of GAD1/Gad1-knockdown or control H520, HT29 and MC38 cell lines. n = 3 per group. b, Analysis of cell numbers in GAD1-knockdown or control H520 (left) and HT29 (right) cell lines (n = 3 per group) in the absence or presence of GABA for 6 d. c, Tumour growth of GAD1-knockdown or control subcutaneous H520 tumours in nude mice. n = 5 per group. Intratumoural injection of GABA was initiated on day 13. d, β-Catenin target gene expression in subcutaneous Gad1-knockdown or control MC38 tumours. n = 3 per group. e, Pearson correlation of GAD1 expression (left) and GABA levels (right) with β-catenin expression in 89 samples from patients with COAD. f, β-Catenin and cyclin D1 protein expression in the indicated GAD1-knockdown or control cancer cell lines. g, mRNA expression of the indicated genes in GAD1-knockdown or control H520 (left) and HT29 (right) cell lines. n = 3 per group. h, β-Catenin and cyclin D1 protein expression in H520 cells incubated with vehicle or 3-MPA (5 μM) for 48 h, followed by treatment with GABA as indicated. i, GAD1, β-catenin and cyclin D1 protein expression in GAD1-knockdown or control H1650 (left) and HT29 (right) cell lines, followed by GABA (50 μM) treatment for 48 h. j,k, Mutant β-catenin(S33Y) or empty vectors (EV) were expressed in GAD1-knockdown or control H520 cells. The indicated proteins were analysed by western blot (j) and cell numbers were examined on day 6 after cells were seeded (k). n = 3 per group. β-Actin was used as a loading control in the western blot analysis. The western blot experiments shown in f and h–j were repeated independently at least twice with similar results. n indicates the number of biological (a–d and k) or technical (g) replicates. Data are mean ± s.d. P values were determined using two-tailed Student’s t-tests (a, b, g and k) and two-way analysis of variance (ANOVA) (c). The correlation coefficient (r) and P values in e were determined using two-tailed Pearson correlation analysis. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. The exact P values are provided in the source data.

Article Snippet: The pcw107-β-catenin (S33Y) vector (Addgene, 64615) and pcw107 empty vector (Addgene, 62511) were gifts from D. Sabatini, J. Doench and K. Wood49. pRRLSIN-β-catenin (∆GSK) (Addgene, 24312) was a gift from R. Nusse50. pRRLSIN empty vector (Addgene, 12252), PAX2 (Addgene, 12260) and VSV-G (Addgene, 12259) were gifts from D. Trono.

Techniques: Derivative Assay, Knockdown, Control, Injection, Targeted Gene Expression, Expressing, Incubation, Mutagenesis, Western Blot, Two Tailed Test

Journal: Science advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling.

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: Fig. 5. BRD9 regulates H3K27me3 levels through the AKT1-EZH2 axis. (A) IB analysis of WCL derived from MDA-MB-231 cells infected with lentivirus of sgGFP or sg- BRD9. (B) IB analysis of WCL derived from MDA-MB-231 cells treated with indicated doses of dBRD9 for 16 hours. (C) IB analysis of WCL derived from MDA-MB-231 cells infected with lentivirus of sgGFP or sgBRD9. Cells were treated with 5 μM GSK126 for 16 hours before harvesting. (D) IB analysis of WCL derived from MDA-MB-231 cells treated with indicated doses of I-BRD9 for 16 hours. (E) IB analysis of WCL derived from MDA-MB-231 cells stably expressing EV or Myr-AKT1 and depleted of BRD9 in- fected with lentivirus sgGFP (−) or sgBRD9 (+). (F) IB analysis of WCL derived from control (sgGFP) or AKT1-depleted MDA-MB-231 cells. Cells were treated with 5 μM I- BRD9 for 16 hours before harvesting. (G) RT-qPCR analysis of mRNA levels of select genes in MDA-MB-231 cells treated with DMSO or 5 μM GSK126 for 48 hours. Data are shown as means ± SD of n = 3 biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.

Article Snippet: Myr- AKT1 (64606) and HA- EZH2 (173717) were purchased from Addgene.

Techniques: Derivative Assay, Infection, Stable Transfection, Expressing, Control, Quantitative RT-PCR

Journal: Science advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling.

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: Fig. 6. Combined treatment of BRD9 and EZH2 inhibitors leads to synergistic growth inhibition of breast cancer. (A) Inhibition of cell viability and dose-response matrixes analyzed by SynergyFinder. MDA-MB-231 cells were treated with the indicated doses of I-BRD9 and GSK126 for 96 hours prior to analysis of cell viability. (B) MDA- MB-231 cells treated with I-BRD9 or GSK126 were subjected to cell proliferation assays. Data are shown as means ± SD of n = 3 biological replicates. ***P < 0.001, two-way ANOVA and Tukey post hoc test. (C) MDA-MB-231 cells treated with I-BRD9 or GSK126 were subjected to colony formation assays. Representative images are shown. (D) Quantification of colonies in (C). Data are shown as means ± SD of n = 3 biological replicates. *P < 0.05 and ***P < 0.001, one-way ANOVA and Tukey post hoc test. (E) Schematic of a mouse xenograft assay to evaluate the antitumor effects of I-BRD9 and GSK126. (F) Tumor growth curve upon treatment of I-BRD9 and GSK126. Data are shown as means ± SEM of n = 6 mice for each group. *P < 0.05, two-way ANOVA and Tukey post hoc test. (G and H) Dissected tumors were weighed. Data are shown as the means ± SEM of n = 6 tumors for each group. *P < 0.05, one-way ANOVA and Tukey post hoc test. (I) Representative images of TUNEL assays in xenograft tumors in (G). (J) Schematic depicting the function of the BRD9-AKT-EZH2 axis in regulating transcription and tumor growth.

Article Snippet: Myr- AKT1 (64606) and HA- EZH2 (173717) were purchased from Addgene.

Techniques: Inhibition, Xenograft Assay, TUNEL Assay

Journal: Cancer cell

Article Title: Dynamic incorporation of histone H3 variants into chromatin is essential for acquisition of aggressive traits and metastatic colonization

doi: 10.1016/j.ccell.2019.08.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For inducible expression Inducer20 GFP, ERK2 D319N and H-RAS G12V, or pCW 57.1 GFP, CHAF1B, CHAF1B shRNA resistant mutant, EGR1, DAXX, HIRA, H3F3A, wild type Sp1, Sp1 AA and Sp1 EE lentivirus were produced by co-transfection of HEK293T cells with plasmids encoding psPAX2 (Addgene plasmid 12260), and pMD2.G (Addgene plasmid 12259) using X-tremeGENE HP (Roche) in accordance with the manufacturer’s protocol.

Techniques: Magnetic Beads, Recombinant, Electron Microscopy, Protease Inhibitor, Transfection, DC Protein Assay, Blocking Assay, Extraction, Mutagenesis, cDNA Synthesis, SYBR Green Assay, Sample Prep, Library Quantification, Purification, Chromatin Immunoprecipitation, DNA Purification, Amplification, Mass Spectrometry, Gene Expression, Binding Assay, Plasmid Preparation, Construct, Control, Software, Real-time Polymerase Chain Reaction, Membrane