stub1  (Santa Cruz Biotechnology)

Journal: Cancer Research

Article Title: A Double-Negative Prostate Cancer Subtype Is Vulnerable to SWI/SNF-Targeting Degrader Molecules

doi: 10.1158/0008-5472.CAN-25-2928

Figure Lengend Snippet: TCF7L2 is a dependency in CRPC-WNT. A, Growth data measured by live-cell imaging (IncuCyte S3) upon transfection of indicated siRNA or plasmid (EV or rescue). Immunoblot of indicated proteins at 72 hours after transfection. GAPDH served as loading control. Data are presented as mean values ± SEM and analyzed using two-way ANOVA. ***, P < 0.001. Data are representative of n = 2 independent experiments. B, Spheroid formation measured by live-cell imaging (IncuCyte SX5) upon transfection of indicated siRNA. Data are presented as mean values ± SEM and analyzed using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of n = 2 independent experiments. C, IHC and staining intensity of TCF7L2 on indicated organoids upon treatment with 1 µmol/L A858 or 1 µmol/L A947 for 24 hours. Violin plot from TCF7L2 staining intensity analyzed using two-way ANOVA. ****, P < 0.0001. Scale bars, 100 µm (MSK-PCa16) and 50 µm (WCM1078). OD, optical density. D, TOPFlash TCF/LEF reporter assay measured after 48 hours upon treatment with indicated drugs in WCM1078 sublines. FOPFlash served as a negative reporter control. Data are presented as mean values ± SEM after normalization to internal Renilla control and analyzed using a paired Student t test. ***, P < 0.001; ****, P < 0.0001. Data are representative of n = 2 independent experiments.

Article Snippet: Chromatin was prepared from two biological replicates of WCM1078 treated with A858 or A947 (1 μmol/L) for 4 hours, and chromatin immunoprecipitation sequencing (ChIP-seq) assays were then performed using an antibody against TCF7L2 (Cell Signaling Technology, cat #2569, RRID: AB_2199816).

Techniques: Live Cell Imaging, Transfection, Plasmid Preparation, Western Blot, Control, Staining, Reporter Assay

Journal: Cancer Research

Article Title: A Double-Negative Prostate Cancer Subtype Is Vulnerable to SWI/SNF-Targeting Degrader Molecules

doi: 10.1158/0008-5472.CAN-25-2928

Figure Lengend Snippet: TCF7L2 regulates proproliferative signatures in CRPC-WNT. A, ChIP-seq read density tornado plots from WCM1078 organoids treated with 1 µmol/L A858 or 1 µmol/L A947 for 4 hours ( n = 2 biological replicates). B, Venn diagram indicating A947 treatment–lost regions from ChIP-seq, ATAC-seq, and RNA-seq data in WCM1078. GSEA was performed from 350 overlapping genes. C, Immunoblot of indicated proteins at indicated time upon treatment with 1 µmol/L A947. GAPDH served as loading control. Data are representative of n = 2 independent experiments. D, Dose–response curves with indicated drugs after measurement of proliferation with CellTiter-Glo 2.0 after 7-day treatment ( n = 2 independent experiments).

Article Snippet: Chromatin was prepared from two biological replicates of WCM1078 treated with A858 or A947 (1 μmol/L) for 4 hours, and chromatin immunoprecipitation sequencing (ChIP-seq) assays were then performed using an antibody against TCF7L2 (Cell Signaling Technology, cat #2569, RRID: AB_2199816).

Techniques: ChIP-sequencing, RNA Sequencing, Western Blot, Control

Journal: microPublication Biology

Article Title: Turnover of missense mutant cytosolic proteins proceeds in the absence of major quality control E3 ligases

doi: 10.17912/micropub.biology.001990

Figure Lengend Snippet: A) Predominant subcellular localization of the QC E3 ligases and reporters used in this study. Dark and light blue represent strong or circumstantial localization evidence in the indicated compartment, respectively, while white indicates no evidence. Data for subcellular localization of E3 ligases was determined through Human Protein Atlas, Uniprot annotations, and existing literature. STUB1, UBE2O, and UBR4 have been reported to localize to both the cytosol and nucleus, whereas UBR5 and HUWE1 are primarily nuclear (Go et al., 2021; Kim et al., 2018; Mashtalir et al., 2014; Tasaki et al., 2013). Subcellular localization of the reporters was determined by microscopy in a previous study (Baker et al., 2025). B) Western blots of indicated E3 ligase KO cells along with the parental HEK293T cells (HEK). * = indicates unspecific band. HUWE1 KO clones were a gift from Yihong Ye’s lab (Xu et al., 2016). C) Schematic of bicistronic fluorescent reporter construct used in D). D) Log 2 (EGFP/DsRed) levels of the missense mutant reporters and corresponding WT in STUB1 KO cells. Three independent KO clones of STUB1 were used. Results from a one-way ANOVA test are shown with the mean and standard deviation (n=3-9, p > 0.05 = ns). E) Schematic of bicistronic fluorescent reporter used in F-J. F) Log 2 (mClover2/RFP) of the mutant reporter and corresponding WT in HEK293T cells. Results from an unpaired t-test are shown with the mean and standard deviation (n=3; **** p < 0.0001). G-I) Log 2 (mClover2/RFP) of the missense mutant reporters CBS L456P, GNMT H177N, and TPMT A80P expressed in (G) UBR4, (H) UBR5 and (I) HUWE1 WT and KO clone(s). Results from an unpaired t-test are shown with the mean and standard deviation (n=3; p > 0.05 = ns, * p < 0.05, ** p <0.01, *** p < 0.001). J) Density plot of the Log 2 (mClover2/RFP) signal of CBS L456P, GNMT H177N, and TPMT A80P in UBE2O WT and KO clones (n=1).

Article Snippet: Primary antibodies used: STUB1 (Cell Signalling 2080S, 1:1000 dilution), UBE2O (Millipore Sigma HPA023605 1:1000), UBR4 (abcam ab86738, 1:1000), UBR5 (Cell Signalling D608Z, 1:1000), HUWE1 (Bethyl A300-486A, 1:1000), g-Tubulin (Sigma T6557, 1:5000), GAPDH (UBC AbLab Mouse, 1:10,000).

Techniques: Mutagenesis, Microscopy, Western Blot, Clone Assay, Construct, Standard Deviation

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A) Binding of KQS-1 to purified human Tregs. Cells were incubated with KQS-1 and analyzed by flow cytometry to determine the proportion of KQS-1–positive cells. Representative gating strategies are shown in . (B, C, D) Raf-1 and ROS dependence of KQS-1–primed Treg responses. Tregs were treated with KQS-1 alone or in combination with the Raf-1 inhibitor GW5074 (10 μM) or the ROS scavenger N-acetylcysteine (NAC, 5 mM). (B) H3K4me3 enrichment at the FOXP3 promoter assessed by ChIP–qPCR and expressed in arbitrary units (a.u.). (C) In vitro suppressive capacity of treated Tregs. The dashed line indicates the mean suppression achieved by KQS-1–treated cells. Representative flow cytometry plots are provided in . (D) TGF-β protein levels in culture supernatants under the indicated conditions. (C) Data are presented as the mean ± SD, with each dot representing an individual donor (n = 10 biologically independent replicates for panel (C)). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. **** P < 0.0001.

Article Snippet: ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.

Techniques: Binding Assay, Purification, Incubation, Flow Cytometry, ChIP-qPCR, In Vitro

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A) ChIP–qPCR analysis of active histone marks at key Treg signature loci. Enrichment of H3K4me3 at the FOXP3 promoter and IL-10 locus, as well as global H3K27ac levels, was assessed in KQS-1–treated and control cells. (B) ATAC-seq volcano plot comparing chromatin accessibility in KQS-1–treated versus untreated Tregs, highlighting newly accessible genomic regions. (C) Genomic annotation of KQS-1–induced accessible regions, categorized as promoters (±3 kb of the transcription start site), enhancers (distal regulatory elements), or intergenic regions. Distribution of accessibility changes across genomic features is shown. (D) Chromatin accessibility at IL-10 and FOXP3 promoter regions. Upper panels show representative ATAC-seq signal tracks; lower panels present quantification of accessibility changes after KQS-1 treatment. (E) Gene ontology (GO) enrichment analysis of genes proximal to KQS-1–induced accessible regions, illustrating enrichment of immune regulatory and T cell–associated pathways. (F) DNA methylation profiling of regulatory regions using the Illumina 450K array. Lollipop plots depict changes in methylation (Δβ) between KQS-1–treated and untreated Tregs, highlighting focal hypomethylation at FOXP3 and IL-10 regulatory CpG sites. ChIP–qPCR data are presented as the mean ± SD (n = 8 biological replicates), with individual points representing replicates. ATAC-seq analysis identified 1,306 newly accessible regions (FDR < 0.05). For genomic distribution analysis, box plots show median and interquartile range (IQR), with individual dots representing peaks. DNA methylation data are shown as mean Δβ with 95% confidence intervals. Statistical significance was determined using paired or unpaired t tests, one-way ANOVA with Tukey’s post hoc test, and the Bonferroni correction for multiple comparisons, as appropriate. ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.

Techniques: ChIP-qPCR, Control, DNA Methylation Assay, Methylation

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: Gain-of-function analysis in Dectin-1-low Tregs after lentiviral transduction with Dectin-1 (Dectin-1 OE) or empty vector control. (A, B) Flow cytometry analysis showing a significant increase in KQS-1 binding (MFI) in Dectin-1 OE cells compared with controls (A), accompanied by restored H3K4me3 epigenetic marks at the FOXP3 promoter region as measured by ChIP–qPCR (B). (C) Suppressive efficiency of Tregs against responder T-cell proliferation, demonstrating enhanced inhibitory capacity upon Dectin-1 restoration. Data are presented as box-and-whisker plots with individual samples shown as dots (n = 5 per group). Statistical significance was determined by an unpaired t test; * P < 0.05, ** P < 0.01.

Article Snippet: ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.

Techniques: Transduction, Plasmid Preparation, Control, Flow Cytometry, Binding Assay, ChIP-qPCR, Whisker Assay