Journal: Cancers

Article Title: Resveratrol and AG490 Overcome Glioblastoma Cells’ Resistance to Monotherapy by Inhibiting JAK2/STAT3 Signalling Pathway

doi: 10.3390/cancers18050794

Figure Lengend Snippet: RES and AG490 suppress STAT3 signaling pathway activation in GBM cells. ( a ) Representative immunofluorescence images showing pSTAT3 (green) and nuclear staining with Hoechst (blue) in LN428 and U251 cells treated with RES, AG490, or RES + AG490. Merged images display the overlay of pSTAT3 and nuclear signals. ( b ) Representative immunocytochemistry results demonstrating pSTAT3 protein expression. ( c ) Representative Western blot results showing total STAT3 and pSTAT3 protein expression in both cells treated with RES, AG490, and RES + AG490. GAPDH serves as the loading control. ( d ) Densitometric quantification of STAT3 and pSTAT3 protein expression levels normalized to GAPDH in LN428 and U251 cells. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 100 µm.

Article Snippet: Afterword, the membranes were blocked with 5% skim milk for 2 h, and washed thrice with Tris-buffered saline (TBS-T, 8 min each), and incubated overnight at 4 °C with primary antibodies, Rabbit polyclonal anti STAT3 (1:1000, Protein Tech, Rosemont, IL, USA 10253-2-AP), Rabbit polyclonal anti pSTAT3 (1:1000, abs118973), Rabbit polyclonal anti BAX (1:1000, Protein Tech, USA 50599-2-lg), Rabbit polyclonal anti BCL-2 (1:1000, Protein Tech, USA 26593-1-AP), and Rabbit polyclonal anti- GAPDH (1:5000, Proteintech, Wuhan, China 10494-1-AP).

Techniques: Activation Assay, Immunofluorescence, Staining, Immunocytochemistry, Expressing, Western Blot, Control

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 2 Effect of DCN on expression and activation of AKT, EGFR, HIF1a, Stat3, and Sp1 in DCN transfected cerebral endothelial cells. (a) Quantitative real-time PCR (top of the gel) and western blot (gel) for AKT are analyzed from the samples (shown in corresponding lanes at the bottom) of mock transfected (control) and three DCN transfected clones (D4, D9, and D10) in five independent experiments. The loading of the samples is normalized with b-actin. (b) Western blot (gel) for phosphorylated AKT (P-AKT) is analyzed from the samples (shown in corresponding lanes at the bottom) of mock transfected (control) and three DCN transfected clones (D4, D9, and D10). Each column represents the mean percentage of phosphorylated AKT out of total AKT of five samples. Total AKT and P-AKT were quantified by histogram values of individual bands subtracting from their back- ground without a band. (c) Effect of AG1478, LY294002, and PTEN transfection are analyzed in western blot from the protein samples of control and DCN transfected MCE clone D10 (MCE-D10) (shown in the corresponding lanes at the top).

Article Snippet: In order to specifically knockdown the expression of either Sp1 or HIF1a or Stat3, DCN synthesizing MCE cells (clone-D10) were transiently transfected with their small interfering RNA (siRNA) vectors (Santa Cruz Biotechnology), as previously described (Santra et al. 2006b,c), followed by SChIP analysis to confirm direct involvement of the expression of Sp1, HIF1a, and Stat3 in DCN-mediated VEGF up-regulation.

Techniques: Expressing, Activation Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control, Clone Assay

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 3 AG1478 and LY294002 treatments inhibit DCN-mediated binding of HIF1a, Stat3, and Sp1 on the VEGF promoter. (a–c) Binding of transcription factors to their specific motifs on the VEGF promoter is analyzed by gel shift assay for the samples from mock transfected MCE cells (MCE-Control), DCN transfected MCE clone D10 (MCE- D10), and MCE-D10 subjected to the different treatments shown in the corresponding lanes. Arrows indicate the retarded probes and un- bound free probes. The transcription factor binding motifs in VEGF promoter are used as their specific oligonucleotide probes in the VEGF promoter, shown at the bottom of the gel. (d) The transcription factors binding specificity is confirmed by super shift analysis. Arrows indicate the retarded probes and super shifted with their specific antibodies (Ab). The samples from DCN transfected MCE cells are pre-incubated with their probes (shown at the bottom of the gel) their specific antibodies (shown in the corresponding lane at the top of the gel).

Article Snippet: In order to specifically knockdown the expression of either Sp1 or HIF1a or Stat3, DCN synthesizing MCE cells (clone-D10) were transiently transfected with their small interfering RNA (siRNA) vectors (Santa Cruz Biotechnology), as previously described (Santra et al. 2006b,c), followed by SChIP analysis to confirm direct involvement of the expression of Sp1, HIF1a, and Stat3 in DCN-mediated VEGF up-regulation.

Techniques: Binding Assay, Gel Shift, Transfection, Control, Incubation

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 4 Quantitative sequential chromatin immunoprecipitation (SChIP) analysis of binding of Sp1, HIF1a, and Stat3 to the VEGF promoter in vivo in DCN synthesizing MCE cells followed by AG1478, LY294002, and PTEN treatment. (a) The locations of the two pairs of primer sets in human VEGF promoter are used to detect the ChIP amplified DNA fragment (190 bp), indicated as filled bar containing Sp1motif (open bar) and DNA fragment (290 bp) indicated as filled bar containing HIF1a (HRE) and Stat3 motifs (open bars). (b) SChIP was performed for the chromatin samples of the corresponding lanes shown in the bottom of the gel by using Sp1, HIF1a, and Stat3 anti- bodies (shown in bottom). Bar graph indicates the fold enrichment that is the relative abundance of DNA fragments at the indicated regions over a control region as quantified by real-time PCR. Migration of PCR products and protein in western blot from SChIP analysis are marked in left. Bar graph at top indicates fold enrichment of SChIP analysis of corresponding samples.

Article Snippet: In order to specifically knockdown the expression of either Sp1 or HIF1a or Stat3, DCN synthesizing MCE cells (clone-D10) were transiently transfected with their small interfering RNA (siRNA) vectors (Santa Cruz Biotechnology), as previously described (Santra et al. 2006b,c), followed by SChIP analysis to confirm direct involvement of the expression of Sp1, HIF1a, and Stat3 in DCN-mediated VEGF up-regulation.

Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Control, Real-time Polymerase Chain Reaction, Migration, Western Blot

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 9 Schematic presentation of pathways of VEGF promoter regu- lation by EGFR/DCN. Activation of Sp1, HIF1a, and Stat3 via DCN/ EGFR/PI3K/AKT and DCN/EGFR/ERK1/2 pathways and eventually activation of VEGF promoter by binding to their consensus motifs into the VEGF promoter are indicated by arrows.

Article Snippet: In order to specifically knockdown the expression of either Sp1 or HIF1a or Stat3, DCN synthesizing MCE cells (clone-D10) were transiently transfected with their small interfering RNA (siRNA) vectors (Santa Cruz Biotechnology), as previously described (Santra et al. 2006b,c), followed by SChIP analysis to confirm direct involvement of the expression of Sp1, HIF1a, and Stat3 in DCN-mediated VEGF up-regulation.

Techniques: Activation Assay, Binding Assay

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: Activated STAT3 is associated with chromatin conformation in HCC. (A) Sub‐clustering of fibroblasts/hepatocytes with respect to STAT3 expression in human HCC scRNA‐seq data ( GSE149614 ). (B) Dot plot showing the percentage of STAT3‐expressed cells and average expression levels of STAT3. (C) Western blotting analysis of cytoplasmic and nuclear fractions from Hep3B and Huh7 cells. Lamin A/C, nuclear marker; α‐tubulin, cytoplasmic marker. Intensity was measured using ImageJ software. Relative intensity was calculated based on fold change, with normalization to ß‐actin intensity. (D) Immunofluorescence staining and quantification of p‐STAT3 (red). Intensity in nuclei was quantified using ImageJ software. Data are plotted as means ± SEM. (E) Heatmap of ChIP‐seq (GSE275343) signals with ±3 kb of unique p‐STAT3 peaks comparing p‐STAT3 enrichment (fold change > 3) between Hep3B and Huh7 cells. (F) Histogram showing the average tag density of p‐STAT3 ChIP‐seq peaks, classified by enrichment. (G) Genomic distribution with respect to p‐STAT3 peaks, annotated by HOMER. (H) Genomic browser snapshot of ChIP‐seq for p‐STAT3 in Hep3B and Huh7 cells. Yellow highlight denotes STAT3 region. * P < 0.05, *** P < 0.001. Abbreviations: HCC, hepatocellular carcinoma; NK, natural killer; scRNA‐seq, single‐cell RNA sequencing; STAT3, signal transducer and activator of transcription 3; p‐STAT3, phosphorylated STAT3; SEM, standard error of the mean; Cyt, cytoplasm; Nuc, nucleus; FC, fold change; ChIP‐seq, chromatin immunoprecipitation sequencing; UTR, untranslated region; TSS, transcription start site; TTS, transcription termination site; chr, chromosome; Ref ., reference genome.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot, Marker, Software, Immunofluorescence, Staining, ChIP-sequencing, RNA Sequencing

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: p‐STAT3‐associated hotspots induce changes in chromatin interactions in HCC. (A) Density plot showing the enrichment of H3K27ac and p‐STAT3 in p‐STAT3 binding regions (GSE275343). (B) Enhancer regions, plotted in order of increasing H3K27ac enrichment in Huh7 compared to Hep3B cells. Enhancers above the inflection point of the curve are defined as Huh7‐enriched enhancer hotspots. (C) Pie chart illustrating the ratio of p‐STAT3 binding in Huh7‐enriched enhancer hotspots. (D) Schematic representation of the definition of p‐STAT3‐associated enhancer hotspots and their linked H3K27ac HiChIP loops (GSE275344). Chromatin regions encompassing multiple enhancers within a proximity of 12.5 kb, as defined by H3K27ac ChIP‐seq, are termed hotspots; regions containing p‐STAT3 binding are referred to as p‐STAT3‐associated enhancer hotspots (GSE275343 and GSE275344). (E) Box plot depicting differences in the interaction strength of p‐STAT3‐associated H3K27ac HiChIP loops between Hep3B and Huh7 cells (GSE275344). (F) Classification of loops based on interaction strength using a 2‐fold change threshold (GSE275344). (G) H3K27ac HiChIP maps (GSE275344) of the 8.5 Mb region of chromosome 1, showing enhancer hotspots associated with active loops in Hep3B and Huh7 cells. Blue lines above figure, lost loops; red lines above figure, gained loops; black dots in figure, loops. (H) Box plot showing the count of loops connected to a single hotspot as a function of type (GSE275344). (I) Depiction of the types and proportions of target loci according to p‐STAT3‐associated hotspots and H3K27ac loops (GSE275343 and GSE275344). (J) Genome browser tracks of the same loci (as in Figure 2G), representing the differential loops linked to p‐STAT3‐associated hotspots between Hep3B and Huh7 cells. FIRE denotes the regions of Huh7 cell gained loops associated with p‐STAT3‐associated hotspots. Yellow box denotes FIRE (GSE275343 and GSE275344). * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: ChIP‐seq, chromatin immunoprecipitation sequencing; STAT3, signal transducer and activator of transcription 3; p‐STAT3, phosphorylated STAT3; HiChIP, high‐throughput chromosome conformation capture followed by immunoprecipitation; chr, chromosome; Ref ., reference genome; FIRE, frequently interacting region.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: Binding Assay, HiChIP, ChIP-sequencing, High Throughput Screening Assay, Immunoprecipitation

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: p‐STAT3‐associated hotspot‐linked loops modify global 3D chromatin conformation. (A) Genome‐wide Hi‐C contact maps ( GSE226215 ) representing all contacts within all chromosomes at 250‐kb resolution, displayed as log2(observed/expected) values. (B) Heatmaps showing the averaged insulation scores around TAD boundaries (GSE275343). (C) Schematic and proportion depicting TAD types – Merged, Splits, Strength‐changed, and Complex – altered in Huh7 cells compared with Hep3B cells ( GSE226215 ). (D) Aggregate TAD analysis of differential TADs between Hep3B and Huh7 cells. Red areas indicate interactions gained in Huh7 cells. (E) Pyramid plots of TADs in Hep3B and Huh7 cells at loci with TAD boundary changes, including Splits and Strength‐changed types. Hep3B and Huh7 cell loops represent cell type‐specific enrichment of H3K27ac HiChIP loops (GSE275344). FIREs refer to the regions of Huh7 cells that gained loops associated with p‐STAT3‐associated hotspots. (F) Genomic track of regions with differential TADs of Splits or Strength‐changed types, corresponding to those shown in Figure 3E. Yellow highlights denote FIREs within the differential TADs. Abbreviation: ChIP‐seq, chromatin immunoprecipitation sequencing; chr, chromosome; TAD, topologically associating domain; CTCF, CCCTC‐binding factor; FC, fold change; FIRE, frequently interacting region; Hi‐C; high‐throughput chromosome conformation capture; HiChIP, high‐throughput chromosome conformation capture followed by immunoprecipitation; STAT3, signal transducer and activator of transcription 3; p‐STAT3, phosphorylated STAT3; Ref ., reference genome.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: Genome Wide, Hi-C, Insulation, HiChIP, ChIP-sequencing, Binding Assay, High Throughput Screening Assay, Immunoprecipitation

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: Identification of highly correlated genes in p‐STAT3‐related FIREs. (A) Proportion of genes located within FIREs connected to p‐STAT3‐associated hotspots. (B‐C) Violin plots showing differential expression of genes located within the identified FIREs across various RNA‐seq datasets. RNA‐seq data in Hep3B and Huh7 cells (GSE275342) (B) and in published HCC patient cohorts ( GSE214846 , GSE77314 , and TCGA‐LIHC) (C). (D) Top schematic: FIRE (orange) with 5’ and 3’ CTCF enrichment (green), p‐STAT3‐associated hotspots (red), and loops gained in Huh7 cells. Lines with arrows denote genes inside and outside the FIRE (GSE275343 and GSE275344). Middle graph: A line plot comparing gene expression correlations within FIREs and between FIRE‐associated genes and adjacent external genes (GSE275342). Bottom graph: Comparison of enrichment of CTCF around FIREs (GSE275343). Bottom schematic: Classification and distribution of correlation patterns. (E) Genome tracking of regions with changes in looping associated with p‐STAT3 hotspots and alterations in TADs. (F) Top: Model of the 3D genome conformation formed by a FIRE, showing looping regions connected to p‐STAT3‐associated hotspots. Middle: Heatmap illustrating correlations of differences in gene expression within the region depicted in Figure 4E, based on fold change data from Hep3B and Huh7 cells (GSE275342). Bottom: Heatmaps depicting correlations of gene expression within p‐STAT3‐associated hotspots and between genes inside and outside a FIRE, using fold change data from normal and tumor samples in various public datasets ( GSE214846 , GSE77314 , and TCGA‐LIHC). Dashed box, genes within the same TAD; yellow highlights, genes located within a FIRE. *** P < 0.001. Abbreviation: 3D, three‐dimensional, ChIP‐seq, chromatin immunoprecipitation sequencing; HCC, hepatocellular carcinoma; HiChIP, high‐throughput chromosome conformation capture followed by immunoprecipitation; STAT3, signal transducer and activator of transcription 3; p‐STAT3, phosphorylated STAT3; FIRE, frequently interacting region; RNA‐seq, RNA sequencing; TCGA‐LIHC, The Cancer Genome Atlas Liver Hepatocellular Carcinoma; TAD, topologically associating domain; CTCF, CCCTC‐binding factor; FC, fold change; chr, chromosome; Ref ., reference genome.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: Quantitative Proteomics, RNA Sequencing, Gene Expression, Comparison, ChIP-sequencing, HiChIP, High Throughput Screening Assay, Immunoprecipitation, Binding Assay

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: Genes regulated by FIREs are associated with tube formation and invasion in HCC. (A) GO analysis of the biological processes associated with genes within FIREs. (B) Network analysis of genes regulated by FIREs. (C) Kaplan‐Meier overall survival plots for HCC patients without or with MVI according to STAT3 status (TCGA‐LIHC). (D) Representative images of tube formation. (E) Spheroid formation by Hep3B and Huh7 cells. The invasive area was quantified at different time points after invasion using ImageJ software. Black arrows indicate invaded areas. (F) GSEA of genes associated with invasion (top) and tube formation (bottom). (G) Heatmap showing invasion‐ and tube formation‐related genes in RNA‐seq profiles of HCC patients ( GSE77314 ). (H) qRT‐PCR analysis of invasion‐ and tube formation‐related gene sets in both HCC cell lines. n.d., not detected; ns, not significant; *** P < 0.001. Abbreviation: FIRE, frequently interacting region; HCC, hepatocellular carcinoma; GO, Gene Ontology; PMA, plasma membrane adhesion molecule; MVI, microvascular invasion; STAT3, signal transducer and activator of transcription 3; GSEA, Gene Set Enrichment Analysis; NES, normalized enrichment score; VCAM1 , vascular cell adhesion molecule 1; ITGA5 , integrin subunit alpha 5; SPINK5 , serine peptidase inhibitor Kazal type 5; MYCN , the proto‐oncogene, basic Helix‐Loop‐Helix (bHLH) transcription factor; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; RNA‐seq, RNA sequencing.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: Software, RNA Sequencing, Quantitative RT-PCR, Clinical Proteomics, Membrane, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: p‐STAT3 remains after sorafenib treatment‐induced changes in genes involved in invasion and tube formation. (A) Western blotting analysis of p‐STAT3 after treatment with IL6 and sorafenib. (B) Heatmap showing changes in the p‐STAT3 ChIP‐seq signal induced by IL6 and sorafenib (GSE275343). (C) Pie chart showing the distribution of p‐STAT3 peaks after sorafenib treatment. (D) Histogram showing the average tag density of p‐STAT3 ChIP‐seq peaks, classified according to IL6 and sorafenib treatment. (E) Venn diagram showing genes in FIREs with stable p‐STAT3. (F) TCGA‐LIHC data showing expression levels of genes in FIREs with stable p‐STAT3 after sorafenib treatment. (G) GO analysis. Blue bar, IL6‐responsive region; pink bar, non‐IL6‐responsive region; purple bar, both. (H) Integrative Genomics Viewer genome browser snapshot showing regions with p‐STAT3 peaks, with and without sorafenib treatment. (I) Relative expression of genes shown in Figure 6H included in FIREs with sustained p‐STAT3. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviation: STAT3, signal transducer and activator of transcription 3; p‐STAT3, phosphorylated STAT3; CTCF, CCCTC‐binding factor; ChIP‐seq, chromatin immunoprecipitation sequencing; Sora, Sorafenib; IL6, Interleukin 6; TCGA‐LIHC, The Cancer Genome Atlas Liver Hepatocellular Carcinoma; CAM, cell adhesion molecule; FIRE, frequently interacting region; chr, chromosome; Ref ., reference genome.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: Western Blot, ChIP-sequencing, Expressing, Binding Assay

Journal: Cancer Communications

Article Title: Coordinated gene expression within sustained STAT3‐associated chromatin conformations contributes to hepatocellular carcinoma progression

doi: 10.1002/cac2.70049

Figure Lengend Snippet: The 3D genome conformation can be targeted for therapeutic manipulation. (A) Single‐cell RNA‐seq analysis with respect to IL6 and IL6R for HCC patients in GSE149614 . (B) Dot plot representing the expression levels of IL6 and IL6R in each cell population ( GSE149614 ). The color of each dot represents the average expression of the gene across the cluster, while the size of each dot indicates the percentage of cells in the cluster expressing IL6 or IL6R (percent expressing). (C) Western blotting analysis showing levels of CTCF and p‐STAT3 upon sorafenib treatment. (D) Depletion of CTCF at protein (left) and mRNA (right) levels in Hep3B and Huh7 cells. (E) Relative expression of genes with CTCF manipulation. (F) qRT‐PCR analysis of CTCF and STAT3 expression following siRNA‐mediated knockdown of CTCF alone or in combination with STAT3 in Huh7 cells. (G) Relative expression of genes within FIREs following knockdown of CTCF alone or combined knockdown of CTCF and STAT3. (H) Model of 3D genome landscape regulated by FIREs with p‐STAT3 in HCC. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviation: 3D, three‐dimensional; STAT3, signal transducer and activator of transcription 3; p‐STAT3, phosphorylated STAT3; HCC, hepatocellular carcinoma; CTCF, CCCTC‐binding factor; IL6, Interleukin 6; IL6R, IL6 receptor; NK, natural killer; M⌽, macrophage; H, hepatocyte; N, normal; T, tumor; FC, fold change; Sora, sorafenib; siRNA, small interfering RNA; siControl, control siRNA; siCTCF, siRNA against CTCF; TAD, topologically associating domain; FIRE, frequently interacting region; qRT‐PCR, quantitative reverse transcription polymerase chain reaction.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membrane (W7031‐270, GenDEPOT, Katy, TX, USA), which were blocked by incubating with 5% skim milk (232100, Beckton, Dicknson and Company, Franklin Lakes, NJ, USA) in PBST (0.1% Tween‐20 in phosphate‐buffered saline), then incubated overnight at 4°C with primary antibodies p‐STAT3 (Tyr705) (9145, Cell Signaling, Danvers, MA, USA), p‐STAT3 (Ser727) (9134, Cell Signaling), STAT3 (4904, Cell Signaling), CTCF (2899, Cell Signaling), α‐tubulin (3873, Cell Signaling), Lamin A/C (sc‐376248, Santa Cruz), and ß‐actin (ab8227, Abcam, Cambridge, UK).

Techniques: RNA Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Knockdown, Binding Assay, Small Interfering RNA, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: The FEBS journal

Article Title: Long non-coding RNA RP11-820 promotes extracellular matrix production via regulating miR-3178/MYOD1 in human trabecular meshwork cells.

doi: 10.1111/febs.15058

Figure Lengend Snippet: Fig. 6. MYOD1 binds STAT3 and activates transcription of ECM genes. (A) Western blot analysis of MYOD1, Fibronectin, Collagen I and Laminin in HTMCs 1 (left) or 2 (right) transfected with the indicated condition. (B) Whole-cell lysates from MYOD1 knockdown HTMCs 1 or scramble cells were immunoprecipitated with anti-MYOD1 antibody. Precipitates were analysed by western blot analysis for MYOD1 and STAT3. (C) Whole-cell lysates from STAT3 knockdown HTMCs 1 or scramble cells were immunoprecipitated with anti-STAT3 antibody. Precipitates were analysed by western blot analysis for MYOD1 and STAT3. (D) mRNA expression of STAT3, collagen I, and fibronectin in STAT3-knockdown or scramble HTMCs 1. (E) mRNA expression of STAT3, collagen I and fibronectin in HTMCs stably expressing STAT3 or control HTMCs 1. (F, G) Western blot analysis of fibronectin, collagen I and laminin in HTMCs 1 (left) or 2 (right) transfected with the indicated condition. The values represent the mean SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Lentiviral expression vector generation and cell line selection To over-express RP11-820 or STAT3, the full-length RP11820 sequence or full-length STAT3 coding sequence was cloned into the PCDH-CMV-MCS-EF1-Puro lentiviral vector (Addgene, Cambridge, MA, USA) between the XbaI and EcoRI restriction sites.

Techniques: Western Blot, Transfection, Knockdown, Immunoprecipitation, Expressing, Stable Transfection, Control

Journal: The FEBS journal

Article Title: Long non-coding RNA RP11-820 promotes extracellular matrix production via regulating miR-3178/MYOD1 in human trabecular meshwork cells.

doi: 10.1111/febs.15058

Figure Lengend Snippet: Fig. 7. Proposed working model. Schematic summarizing our proposed model for lncRNA-RP11-820 regulates ECM genes in HTMCs. lncRNA-RP11-820 directly adsorb miR-3178 as a sponge, through which upregulate the expression of MYOD1. Importantly, MYOD1 can transcriptionally bind to the promoter region of ECM gene, leading to increased ECM protein deposition. Furthermore, MYOD1 can bind STAT3 to form a transcription complex, and transcriptionally activates ECM genes expression in HTMCs.

Article Snippet: Lentiviral expression vector generation and cell line selection To over-express RP11-820 or STAT3, the full-length RP11820 sequence or full-length STAT3 coding sequence was cloned into the PCDH-CMV-MCS-EF1-Puro lentiviral vector (Addgene, Cambridge, MA, USA) between the XbaI and EcoRI restriction sites.

Techniques: Expressing

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control