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: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) GSK3β for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Transfection, Control, Western Blot, Expressing, Infection, Staining, Fluorescence

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: Wild-type MEFs were transfected with non-targeting (CONsi) or GSK3β-specific (GSK3βsi) siRNAs (100nM) for 48hrs. A ) Western blotting for GSK3β confirmed depletion of the protein in GSK3βsi-transfected MEFs. GAPDH was used as a loading control. B ) CONsi and GSK3βsi-transfected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of CRC measurements using Calcium Green-5N in digitonin-permeabilized CONsi and GSK3βsi-transfected MEFs. D ) Quantification of CRC data in the CONsi and GSK3βsi-transfected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. CONsi.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Transfection, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: A ) Upper panels, Western blotting for GSK3β, lactate dehydrogenase (LDH, cytosolic), and succinate dehydrogenase (SDHB, mitochondrial) in cardiac mitochondrial and cytosolic subfractions. Lower panels, Western blotting for GSK3β, VDAC (membrane), and CypD (soluble) in alkali extracted membrane and soluble fractions of cardiac mitochondria. B ) Upper panels, recombinant GST-rat GSK3β was incubated with His-tagged human CypD, purified on Co 2+ -agarose column, and the complexes Western blotted for GST and CypD. Lower panels, recombinant GST-GSK3β was incubated with His-tagged human CypD in kinase buffer supplemented with ATP, purified on Co 2+ -agarose column, and the complexes Western blotted for phosphoserine/threonine, GST and CypD. C ) Mass analysis of phosphorylated recombinant CypD demonstrating the presence of two phosphorylation sites. D ) Example of MS-MS spectra indicating S101 as a putative GSK3β phosphorylation site on CypD. E ) Amino acid sequence of the recombinant His-tagged human CypD depicting canonical GSK3β consensus sites (red), the putative phosphorylation sites identified in our analyses (blue), and a site previously hypothesized to be a key GSK3β phosphorylation residue (purple). F ) Surface representations of human CypD illustrating the positions of the various putative phosphorylation sites and their proximity to the catalytic CsA-binding domain (CsA-BD).

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Western Blot, Membrane, Recombinant, Incubation, Purification, Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Residue, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: Wild-type MEFs were infected with adenoviruses encoding βGal (control) or mitochondrially-targeted HA-tagged WT-GSK3β (miWT-3β) or DN-GSK3β (miDN-3β) for 48hrs. A ) Western blotting for HA and GSK3β confirmed equivalent expression of the two GSK3β proteins. GAPDH was used as a loading control. B ) Representative images of immunocytochemistry performed on MEFs infected with miWT-3β stained for the HA tag confirmed mitochondrial localization of the protein. Mitochondria were visualized by staining for CypD. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, miWT-3β and miDN-3β-infected MEFs. Cells were treated with 2.5μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, miWT-3β and miDN-3β-infected MEFs. E ) βGal, miWT-3β and miDN-3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. Each individual point represents one independent cell isolate. Bar represents the mean.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Infection, Control, Western Blot, Expressing, Immunocytochemistry, Staining, Fluorescence

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: A) Cardiac mitochondria were exposed to increasing concentrations of proteinase K and then Western blotted for hexokinase-2 (HK2, mitochondrial surface), apoptosis-inducing factor (intermembrane space), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2, inner membrane), and CypD (matrix). B) MEFs were infected with adenoviruses encoding βGal, WT-GSK3β and/or FLAG-tagged CypD for 48hrs. CypD was immunoprecipitated using an anti-FLAG antibody and the complexes Western blotted for GSK3β and FLAG. C) CypD KO MEFs were infected with adenoviruses encoding βGal, WT-GSK3β for 48hrs. Western blotting for GSK3β confirmed overexpression of GSK3β. GAPDH was used as a loading control. D) βGal and WT-GSK3β-infected CypD KO MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. E) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, and WT-GSK3β -infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. F) Quantification of CRC data in the βGal and WT-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Western Blot, Membrane, Infection, Immunoprecipitation, Over Expression, Control, Staining, Fluorescence