Journal: Molecular Metabolism

Article Title: Identification of a chromatin-bound ERRα interactome network in mouse liver

doi: 10.1016/j.molmet.2024.101925

Figure Lengend Snippet: Liver ERRα and HCFC1 chromatin association. (A) Venn diagram illustrating the overlap in genes found targeted by ERRα (GSE43638) and HCFC1 (GSE115767) within ± 20 kb of gene TSSs from ChIP-seq data in mouse liver. (B) Heatmap representing the binding signal intensities of resulting peak regions from ERRα (GSE43638) and HCFC1 (GSE115767) peak intersections based on peaks found ± 20 kb of gene TSSs. (C) Genome annotations of peak locations for ERRα-specific, HCFC1-specific, and ERRα and HCFC1overlapping peaks (common) identified in (B) expressed as a percentage of the total peaks. (D) Distribution of peak positions for the 601 identified ERRα and HCFC1 overlapping peak regions in (B) relative to gene TSSs. (E) HOMER de novo motif analysis of peak lists in (B). (F) ChIP-seq read density graphs showing the distribution of HCFC1 peak summits relative to ERRα peak summits for the 601 shared regions identified in (B). (G) ChIP-seq tracks showing ERRα and HCFC1 colocalization at the indicated genes with the presence of an HCFC1 peak found directly upstream or downstream that of ERRα. (H) Top, schematic illustrating the co-occupancy of ERRα and HCFC1 at nuclear-encoded OXPHOS genes. The number of co-bound OXPHOS genes for each respiratory complex is shown and the proportion it represents among all nuclear-encoded genes. Bottom, heatmap showing mouse liver ChIP-qPCR enrichment values for ERRα and HCFC1 binding at OXPHOS co-targeted genes, validating their simultaneous occupancy at common sites. Control regions were used to validate ERRα- and HCFC1-uniquely bound regions. The data shown are from one experiment representative of two independent experiments. (I) Heatmap of ChIP-seq binding signal intensities for HCFC1 in Hepa 1–6 cells ± siRNA-mediated ERRα or HCFC1 knockdown at the 601 ERRα and HCFC1 co-occupied regions identified in mouse liver from (B). (J) HCFC1 ChIP-qPCR experiments in Hepa 1–6 cells ± siRNA-mediated ERRα or HCFC1 knockdown at established ERRα and HCFC1 co-bound sites as well as HCFC1- and ERRα-specific sites (n = 3). Data (J) are presented as means ± SEM, ∗p < 0.05, unpaired two-tailed Student's t test. See also Suppl. Fig. 4 .

Article Snippet: The day before nuclear lysates were prepared, Protein G Dynabeads (50 μL, 10008D, Thermo Fisher Scientific) were washed with 1 ml of cold 1X PBS-BSA (1X PBS with 0.5% BSA) then subsequently incubated with 500 μl of cold 1X PBS-BSA containing either 3 μL of ERRα antibody (homemade, designed against N-terminal amino acid residues 1–74 of human ERRα), 10 μL of HCFC1 antibody (A301-399 A, Bethyl) or 1 μg of rabbit IgG (10500C, Invitrogen) and left to rotate overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, ChIP-qPCR, Control, Knockdown, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Identification of a chromatin-bound ERRα interactome network in mouse liver

doi: 10.1016/j.molmet.2024.101925

Figure Lengend Snippet: ERRα transcriptional regulation of OXPHOS genes is HCFC1-dependent. (A,B) Evaluation of the effectiveness of a pool of siRNAs against ERRα and/or HCFC1 in Hepa 1–6 cells at the protein (n = 2) (A) and mRNA (n = 4) (B) level by Western blot and RT-qPCR analyses, respectively. (C) GSEA analysis of Hepa 1–6 transcriptomes upon siRNA-mediated ERRα and/or HCFC1 knockdown (n = 3). Significantly enriched gene sets with an FDR < 0.25 are shown. Bars represent the normalized enrichment score (NES): red and blue bars signify that the gene set is upregulated and downregulated upon the indicated knockdown, respectively. (D) Heatmap summarizing the common GSEA hallmark terms found independently modulated by the loss of ERRα or HCFC1 alone in (C). (E,F) GSEA enrichment plots for the significantly enriched hallmark term OXPHOS (E) and cell cycle-related term G2M_CHECKPOINT (F) shown in (D). (G) RT-qPCR examination of the effects of a pool of siRNAs against ERRα and/or HCFC1 in Hepa 1–6 cells on the expression of co-targeted OXPHOS genes (n = 4). (H) Basal and maximal. mitochondrial respiration rates of Hepa 1–6 cells treated with siRNAs against ERRα and/or HCFC1 (n = 4) measured using a Seahorse Flux Analyzer. (I) Assessment of cell proliferation after a 72 h siRNA-mediated knockdown of ERRα and/or HCFC1 in Hepa 1–6 cells (n = 6) expressed as a percentage relative to the control knockdown (siC). Data (B, G-I) are presented as means ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA with Tukey's post-hoc analysis.

Article Snippet: The day before nuclear lysates were prepared, Protein G Dynabeads (50 μL, 10008D, Thermo Fisher Scientific) were washed with 1 ml of cold 1X PBS-BSA (1X PBS with 0.5% BSA) then subsequently incubated with 500 μl of cold 1X PBS-BSA containing either 3 μL of ERRα antibody (homemade, designed against N-terminal amino acid residues 1–74 of human ERRα), 10 μL of HCFC1 antibody (A301-399 A, Bethyl) or 1 μg of rabbit IgG (10500C, Invitrogen) and left to rotate overnight at 4 °C.

Techniques: Western Blot, Quantitative RT-PCR, Knockdown, Expressing, Control

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Co-immunoprecipitation for CUX1 in K562 cells was followed by mass spectrometry (n=2 biological replicates). The heatmap indicates BAF members ranked by the mean label-free quantification fold enrichment compared to IgG controls. (B) Representative co-immunoprecipitation followed by immunoblot in K562 (n=2 biological replicates). (C) K562 CUX1 and SMARCA4 ChIP-seq overlap (n=2 biological replicates, IDR<0.05). (D) Enriched motifs84 at CUX1 and SMARCA4 co-occupied sites. (E) Overlap of SMARCA4 peaks (n=2 biological replicates, IDR<0.05) in gHPRT and gCUX1 K562 cells. (F) Heatmaps showing overlap between CUX1-dependent or CUX1-independent SMARCA4 sites with CUX1. The values are normalized ChIP-seq reads (RPKM). The direct model represents CUX1 recruitment of SMARCA4. The indirect model represents SMARCA4 sites bound but not recruited by CUX1. Example genome snapshots for each category are shown (G).85 (H) Distance to the nearest transcription start site (TSS) of CUX1-recruited and non-CUX1-recruited SMARCA4 sites and hematopoietic TF occupancy (I).

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Western Blot, ChIP-sequencing

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Volcano plot comparing ATAC-seq signal in gCUX1 vs. gHPRT K562 cells (n=2 biological replicates). Significance calculated by csaw.41 Top enriched motifs for the significant down sites are shown. (B) H3K27ac ChIP-seq reads (n=2 biological replicates) at significantly down, up and non-significant ATAC sites. (C) CUX1 and SMARCA4 occupancy at down (n=933) vs. CUX1-independent ATAC sites (n=14,256). ATAC-seq signal from gHPRT and gCUX1 cells for CUX1 and SMARCA4 co-occupied sites (D), CUX1-recruited SMARCA4 sites (E), and SMARCA4 sites bound but not recruited by CUX1 (F). Significance for (B-F) calculated by two-sided Wilcoxon rank-sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: ChIP-sequencing

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Overlap of CUX1 and SMARCA4 CUT&RUN peaks in primary human CD34+ HSPCs (n=2 biological replicates, IDR<0.05). (B) Genome-wide correlation of CUX1 and SMARCA4 CUT&RUN signals with histone marks from Roadmap Epigenomics.44 All pairwise correlations have p<0.001. (C) CUX1 and SMARCA4 peaks absolute distance (log2 transformed) to the nearest TSS. The dash line indicates 2 Kb. (D) Top GO terms for TSS-proximal and -distal CUX1/SMARCA4 co-bound sites (Bonferroni corrected p-value<0.05).45,46 (E) Volcano plot of ATAC-seq changes in gCUX1 and gHPRT CD34+ HSPCs (n=2 biological replicates). Significance calculated by csaw.41 Top motifs for the down sites are shown. (F) Normalized ATAC reads at genome-wide CUX1-bound enhancers (n=3,902) and a randomly sampled, size-matched list of enhancers not bound by CUX1 (top). Normalized ATAC reads at CUX1-bound enhancers (n=3,902) comparing the control gHPRT and gCUX1 conditions (bottom). (G) Normalized CUT&RUN reads of CUX1 and SMARCA4 in CD34+ HSPC at down vs. CUX1-independent ATAC sites. Significance for (F) and (G) is by two-sided Wilcoxon rank-sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Genome Wide, Transformation Assay, Control

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) ATAC-seq accessibility for gHPRT and gCUX1 CD34+ HSPCs at distal 3D chromatin contact points looped to CUX1-bound promoters from published CD34+ HSPC Hi-C data.59 (B) IGV snapshot of CUX1 binding at the promoter of KIT and the reduced accessibility of multiple enhancers looped to the promoter. Enhancer and promotor annotations are from Roadmap Epigenomics.44 (C) Integration of CD34+ HSPC ATAC-seq and RNA-seq (n=2 biological replicates).13 Scatterplot shows the RNA log2FC vs. ATAC-seq log2FC for 406 DEGs (FDR<0.1, |log2FC|>0.75). Enriched GO terms related to HSPC lineage commitment are shown.86 (D) Log2FC of ATAC-seq signal comparing CD34+ HSPC gCUX1 vs. gHPRT cells at the hematopoietic cell-type specific enhancers from the VISION database87 (9,657 myeloid enhancers, 11,653 erythroid enhancers, 15,323 lymphoid enhancers), and 10,000 randomly sampled non-cell type specific enhancers. (E) Performance score of cell fate prediction using the published murine HSPC scRNA-seq.68 From left to right: positive control is the top 2,000 genes with the highest cell-cell variation; negative controls are a randomly sampled gene set (n=1,000) and curated list of mouse TFs (n=1,636);88 CUX1-bound genes from human CD34+ HSPC CUT&RUN (n=6,758); overlap of CUX1-bound and differentially-expressed upon CUX1 knockdown in CD34+ HSPC (n=923). Equivalent gene sets were tested for PU.1 and RUNX1 as benchmarks (n = 336 and 325).69–72 For all gene sets larger than 1,000, 50 bootstraps were performed to sample for 1,000 genes. Logistic regression and deep neural network were used to construct the classifier. Significance for (A), (D), (E) is by two-sided Wilcoxon rank sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Hi-C, Binding Assay, RNA Sequencing, Positive Control, Knockdown, Construct

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: Key resources table

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Purification, Mass Spectrometry, Software

Journal: Genes & Development

Article Title: Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization

doi: 10.1101/gad.263327.115

Figure Lengend Snippet: Prrx1b–Hgf signaling regulates pancreatic cancer cell invasion. ( A ) Prrx1a and Prrx1b target genes identified by ChIP-seq. Genes of enriched pathways (KEGG) are indicated. ( B ) Relative mHgf gene expression in control and Prrx1a-overexpressing and Prrx1b-overexpressing KP fl CY cells ( left ) and control and Prrx1a and Prrx1b knockdown KPC2 cells ( right ). (*) P < 0.05; mean ± standard deviation (SD). ( C ) Invasion assay in control and Prrx1a and Prrx1b knockdown cells using mrHGF. ( D ) Double IHC staining for PRRX1B (brown) and mHGF (blue) in KP fl CY pancreatic tumors (orthotopic transplantation model). Bars, 50 μm. ( E ) Quantification of secreted mHGF protein by ELISA. n = 3; mean ± SEM. ( F ) Secreted mHGF protein in the supernatant of control and Prrx1a/b knockdown KPC2 cells. n = 5; mean ± SEM.

Article Snippet: The human pancreatic cancer cell line Panc1 was purchased from American Type Culture Collection and maintained in Dulbecco's modified Eagle medium (DMEM) (Sigma Chemical Co.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. mrHGF (10 ng/mL; R&D Systems, 2207-HG ) or 10ng/mL human recombinant HGF (R&D Systems, 294-HG) was used for HGF stimulation assays.

Techniques: ChIP-sequencing, Gene Expression, Control, Knockdown, Standard Deviation, Invasion Assay, Immunohistochemistry, Transplantation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A dual mechanism of enhancer activation by FOXA pioneer factors induces endodermal organ fates

doi: 10.1101/2020.08.28.263020

Figure Lengend Snippet: ( A and B) Schematic of stepwise differentiation of hESCs to hepatic progenitors (HP) ( A ) and induced human pluripotent stem cells (iPSC) into alveolosphere organoids (ALV) ( B ). AFG, anteriorized foregut. Select growth factors for hepatic ( A ) and alveolar ( B ) lineage induction are indicated. FGF2, fibroblast growth factor 2; BMP4, bone morphogenic protein 4; CHIR, CHIR99021; RA, retinoic acid. ( C ) Heatmap showing density of FOXA1 and FOXA2 ChIP-seq reads at hepatic enhancers in GT and HP. Heatmap is centered on FOXA1 and FOXA2 peaks, respectively, and spans 5 kb. Hepatic enhancers are classified based on temporal pattern of FOXA1 and FOXA2 occupancy. ( D ) Heatmap showing density of FOXA1 ChIP-seq reads at hepatic enhancers in AFG and ALV. Heatmap is centered on FOXA1 peaks and spans 5 kb. Alveolar enhancers are classified based on temporal pattern of FOXA1 occupancy. ( E ) Genome browser snapshots showing FOXA1 and FOXA2 ChIP-seq signal at a class I hepatic enhancer near AAT and a class II hepatic enhancers near CEBPA in GT and HP. ( F ) Genome browser snapshots showing FOXA1 ChIP-seq signal at a class I alveolar enhancer near SOX2 and a class II alveolar enhancer near NKX2.1 in AFG and ALV. ( G and H ) Enriched de novo transcription factor (TF) binding motifs at class I against a background of class II enhancers and vice versa for hepatic ( G ) and alveolar enhancers ( H ). Fisher’s exact test, 1-sided, corrected for multiple comparisons. All ChIP-seq experiments, n = 2 replicates from independent differentiations. See also and , Tables S5 and S6.

Article Snippet: ‘‘CBRa’’ medium consists of cSFDM base supplemented with 3 μM CHIR99021 (Tocris), 10 ng/mL recombinant human BMP4 (rhBMP4, R&D Systems), and 100 nM retinoic acid (RA, Sigma), as described ( ).

Techniques: ChIP-sequencing, Binding Assay

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 1. TGFβ pathway signature increases in TNBC cells upon anchorage-independent culture and androgen receptor and SMAD3 are predicted up- stream regulators. (A) Metacore pathway analysis of BT549 microarray data showing SMAD3 connecting with other genes altered under anchorage- independent culture for 24 hours. Original data set from Barton et al (2015) (GEO record GSE95472). (B) GSEA pathway enrichment analysis showing changes in the TGFβ pathway in BT549 cells cultured in attached or under anchorage-independent culture for 24 hours. (C) Heatmap of significantly altered genes associated with the TGFβ pathway in BT549 cells grown in attached versus suspended culture conditions for 24 hours (n = 4). Gene list from KEGG database on TGFβ Signaling Pathway. Red asterisk: genes associated with the canonical TGFβ signaling pathway. (D) qRT-PCR for ca- nonical TGFβ signaling pathway gene expression in attached versus under anchorage-independent culture conditions at 48 hours in SUM159PT and BT549 cell lines (n = 6). Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Microarray, Cell Culture, Quantitative RT-PCR, Gene Expression

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 2. Anchorage-independent conditions increase TGFβ1 expression and secretion and nuclear pSmad3 localization and activity. (A) RPPA data showing changes in protein in BT549 cells grown in attached versus under anchorage-independent culture conditions for 24, 48, and 72 hours with biological triplicates at each condition and timepoint shown. Red, high relative expression levels; black, intermediate relative expression levels; green, low relative expression levels. Full panel of RPPA data in (30). (B) ELISA measuring changes in TGFβ1 in conditioned medium from three TNBC lines cultured in attached versus suspended conditions for 24 and 48 hours normalized to total live cell count as determined by trypan blue. (C) IHC and quantification of percentage of pixels corresponding to nuclear DAB staining for pSmad3 in MDA-MB-453, SUM159PT, and BT549 cells in at- tached versus suspended culture conditions for 24 hours. (D) 3TP-lux TGFβ reporter-linked luciferase activity normalized to renilla in SUM159PT cells grown in attached versus suspended culture. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Counting, Staining, Luciferase

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 3. Exogenous TGFβ increases AR protein levels and TGFβ inhibition decreases AR and cell viability in anchorage-independent conditions. (A) Western blot for pSmad2, Smad2, Smad3, ID1, AR, GR, and GAPDH in the SUM159PT cell line treated with recombinant TGFβ1 (10 ng/mL) over a time course of 24 hours. (B) qRT-PCR for AR in SUM159 and BT549 cells grown in attached conditions for 48 hours ± LY2109761 (10 µM). (C) Western blot for AR was performed on MDA-MB-453, SUM159PT, and BT549 cell lines grown in attached or suspended conditions for 48 hours ± LY2109761 (10 µM). (D) Western Blot to analyze the apoptosis marker cleaved-PARP in SUM159PT and BT549 cells in attached or suspended conditions for 48 hours ± LY2109761 or LY2157299 (10 µM). Densitometry values calculated as (cleaved-PARP/PARP)/GAPDH. (E) Trypan blue assay for percentage of dead cells in SUM159PT and BT549 cells in attached or suspended conditions for 48 hours ± LY2109761 or LY2157299 (10 µM). Western blot quantifica- tions performed by ratio of protein of interest to loading control and normalized to control. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Inhibition, Western Blot, Recombinant, Quantitative RT-PCR, Marker, Control

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 4. Clinical data suggests a high AR and TGFB3 is associated with worse outcome. (A) Correlation analysis on the co-expression of AR and the TGFβ ligand TGFB3 in TNBC patients in the TCGA-BRCA (n = 123) and SCAN-B (n = 143) cohorts. (B) GSEA analysis on TGFB3 relating to the androgen response gene set from the HCI-009 PDX. (C) KMplotter was used to stratify basal BC patients stratified into ARhigh (top) or ARlow (bottom) populations. TGFB3 expression was then inter- rogated based on overall survival (OS, n = 241) and distant metastasis free survival (DMFS, n = 232)

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 5. AR ChIP-seq peaks are identified near SMAD3 and TGFB1. (A) Heatmaps show that differential AR genome binding in attached and sus- pended conditions with vehicle, DHT, and DHT+Enza treatments. (B) Heatmaps display the signal at differential DHT-induced AR binding sites that show a 2-fold or greater enrichment in the suspended condition. (C) Gene ontology biological processes found nearby AR bound sites that are more than 2-fold enriched in suspended cells using GREAT (63). (D) Genes involved in focal adhesion assembly that were identified as being 2-fold en- riched in under anchorage-independent culture by AR ChIP-seq. (E) AR ChIP-seq peaks identified near SMAD3 are shown. The scale bar at the top is labeled for each loci to indicate the relative distance from the canonical SMAD3 TSS along with the respective gene annotations. For each region, the AR ChIP-seq signal for each treatment condition in attached (black) and suspended (red) are shown. The peaks at each loci are scaled to the same height for accurate comparison but are not directly comparable between loci due to differences in ChIP-seq signal intensity. The chromosomal coord- inates of the area covered by each peak are shown below each region. (F) Browser tracks show AR binding near the TGFβ1 promoter. AR ChIP-seq signal within a 2000 bp window covering 500 bp upstream of the TGFβ1 TSS and 1500 bp into the 5′ UTR and first exon is shown. ChIP-seq signal is scaled to the same height for comparison and represents each treatment condition in attached (black) and suspended (red), with the chromosomal coordinates included at the bottom for reference.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: ChIP-sequencing, Binding Assay, Labeling, Comparison

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 6. SUM159PT cells are more sensitive to a combined treatment with Enza and TGFβ inhibitor than either drug alone, particularly after anchorage-independent culture. (A) SUM159PT cells were grown in triplicate in 6-well plates in attached conditions for 72 hours with either vehicle, LY1 (10 μM), Enza (40 μM), or both, followed by crystal violet assay. (B) SUM159PT cells were grown in triplicate in 6-well plates in attached conditions for 72 hours with the same treatments, followed by washout of drug and an additional 72 hours in culture, then crystal violet assay was performed. (C) SUM159PT cells were grown in under anchorage-independent culture on poly-hema coated plates for 48 hours with the treatments mentioned above, followed by replating of cells in attached conditions for an additional 48 hours. Crystal violet assay was performed after. Mean ± SD; *P < .05; **P < .01; ***P < .001. ****P < .0001.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Crystal Violet Assay

Journal: Endocrinology

Article Title: A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

doi: 10.1210/endocr/bqaa226

Figure Lengend Snippet: Figure 7. Proposed model of a positive feedback loop whereby TGFβ signaling increases AR expression, and AR increases core TGFβ pathway components.

Article Snippet: Cells were treated with 10 ng/mL of recombinant human TGFβ1 (R&D Systems).

Techniques: Expressing

Journal: Nature Communications

Article Title: The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis

doi: 10.1038/s41467-023-44053-w

Figure Lengend Snippet: a SF were left untreated or stimulated with TNF ( n = 21), LPS ( n = 9), poly I:C (PIC) ( n = 9), TGFβ ( n = 9), IL1β ( n = 9), or bacterial lipoprotein (bLP)( n = 3) for 24 h. Expression of HOTAIR was measured by qPCR. Unstimulated samples were set to 1. Mean +/− standard deviation is shown, one sample t test (all but IL1β). IL1β: median and range, one sample Wilcoxon. b CAGE analysis of enhancers/promoters at the HOTAIR locus in SF from knees ( n = 2) in basal conditions and after stimulation with TNF. After TNF stimulation several active promoters (light blue bars) and the enhancer (pink bar) disappear in knee SF. c Correlation between TNF and HOTAIR expression measured by qPCR in RA synovial tissues. Pearson correlation, one-sided. d Expression of Hotair was measured by qPCR in knee and ankle SF isolated from wildtype (WT) ( n = 14) and TNF transgenic (Tg197) ( n = 13) C57BL/6 mice. Median and range is shown. Mann–Whitney test. e ChIP sequencing of H3K27me3 marks in SF silenced for HOTAIR ( n = 3) and control SF ( n = 3) was performed 48 h after transfection. TSS Transcription start site. SF synovial fibroblasts, RA rheumatoid arthritis, OA osteoarthritis. Not reported p -values on the figure were not significant.

Article Snippet: SF were stimulated with human recombinant TNF (10 ng/ml; R&D Systems), human recombinant IL1β (1 ng/ml; R&D Systems), lipopolysaccaride (LPS) from Escherichia coli J5 (100 ng/ml; List Biological Laboratories), polyI-C (PIC) (10 μg/ml; InvivoGen), bacterial lipopeptide (bLP) palmitoyl-3-cysteine-serine-lysine-4 (300 ng/ml; InvivoGen) or human recombinant TGFβ (10 ng/ml; R&D Systems) for 24 h. For DNA demethylation, SF were treated daily with complete DMEM 10%FCS with 1 μM 5-azacytidine (Sigma Aldrich) for 5 days.

Techniques: Expressing, Standard Deviation, Isolation, Transgenic Assay, MANN-WHITNEY, ChIP-sequencing, Control, Transfection