non-pou domain-containing octamer-binding protein (nono) antibody  (Novus Biologicals)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU interacts with and induces cytoplasmic retention of NONO in gastric cancer cells. a , Coomassie bright blue staining (left panel) and Venn diagram (right panel) showing the differential proteins pulled down by biotin-labeled sense (S) or antisense (AS) probe targeting junction sites of circ-hnRNPU from the lysates of AGS cells, and overlapping analysis of proteins identified by mass spectrometry (MS) with established RBP from RBPDB ( http://rbpdb.ccbr.utoronto.ca ) and c-Myc-binding proteins derived from BioGRID and IntAct ( https://www.ebi.ac.uk/intact ) databases. b , Western blot (upper panel) and RT-PCR (lower panel) assays indicating the NONO protein or circ-hnRNPU pulled down by biotin-labeled S or AS probes targeting junction site of endogenous circ-hnRNPU from lysates of AGS cells. c , RIP (upper panel) and western blot (lower panel) assays using NONO antibody showing the interaction of NONO with circ-hnRNPU or hnRNPU mRNA in MKN-45 and AGS cells stably transfected with empty vector (mock) or circ-hnRNPU . d , RNA EMSA determining the interaction between recombinant GST-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU , with or without competition using an excess of unlabeled circular probe. e , Schematic diagram of NONO truncations (left panel) and in vitro binding assay (middle and right panels) depicting the recovered circ-hnRNPU levels detected by RT-PCR after incubation with full-length or truncation forms of GST-tagged or Flag-tagged recombinant NONO protein validated by western blot. f – h , Representative images and quantification of dual RNA-FISH and immunofluorescence ( f ), real-time qRT-PCR ( g , normalized to β-actin ), and western blot ( h ) assays indicated the localization of circ-hnRNPU and NONO (arrowheads), transcript and protein levels of NONO in MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU , lin-hnRNPU , scramble shRNA (sh-Scb), or sh- circ-hnRNPU . Scale bar: 10 μm. Student’s t test compared the difference in f and g . ** P < 0.01. Data are representative of three independent experiments in b - h

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Staining, Labeling, Mass Spectrometry, Binding Assay, Derivative Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Plasmid Preparation, Recombinant, In Vitro, Incubation, Immunofluorescence, Quantitative RT-PCR, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits interaction of NONO with c-Myc in gastric cancer cells. a, Immunofluorescence assay showing the localization of NONO and c-Myc protein (arrowheads) in AGS cells stably transfected with empty vector (mock), NONO , or c-Myc . Scale bar: 10 μm. b , Schematic diagram, co-IP and western blot assays indicating the interaction between full-length or truncations of recombinant GST-tagged c-Myc and MBP-tagged NONO proteins. c , Co-IP and western blot assays revealing the direct interaction between recombinant MBP-tagged NONO and GST-tagged c-Myc proteins, with or without treatment by in vitro generated circ-hnRNPU . d , BiFC assay showing the interaction between NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock, circ-hnRNPU , scramble shRNA (sh-Scb), or sh-circ-hnRNPU #1. Scale bar: 10 μm. e , Representative images (left panel) and quantification (right panel) of immunofluorescence assay revealing the co-localization of NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock or circ-hnRNPU . Scale bar: 10 μm. f , Immunofluorescence (lower left panel), RIP and western blot (lower right panel) assays showing the localization (arrowheads) and interaction of NONO with circ-hnRNPU or c-Myc protein in AGS cells transfected with Flag-tagged wild-type (WT) or NLS-mutant (mNLS) form of NONO as indicated (upper panel). Student’s t test compared the difference in e . ** P < 0.01. Data are representative of three independent experiments in a - f

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Recombinant, In Vitro, Generated, Bimolecular Fluorescence Complementation Assay, shRNA, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits dual NONO activity in regulating c-Myc transactivation and mRNA stabilization. a and b , Dual-luciferase assay using a reporter containing three canonical c-Myc binding sites ( a ), ChIP and qPCR (b, normalized to input) assays indicating c-Myc transactivation and enrichment on target gene promoters in MKN-45, AGS, and HeLa cells stably transfected with empty vector (mock) or circ-hnRNPU , and those co-transfected with NONO or c-Myc ( n = 5). c and d , Dual-luciferase assay ( c ), western blot (d, left panel), and real-time qRT-PCR (d, normalized to β-actin , right panel) assays showing promoter activity or expression of GALNT6 and MGAT1 , as well as circ-hnRNPU or downstream targets ( FN1 and GLUT1 ) levels in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with NONO or c-Myc . e , Venn diagram (left pane) and CLIP-seq peak (middle panel) indicating comprehensive analysis of 112 altered genes in RNA-seq and NONO-binding targets (GSE90650). RIP and real-time qRT-PCR assays (right panel) revealing endogenous NONO binding to 3'-UTR of hnRNPU , GALNT2 , and GALNT6 in AGS cells. f and g , Dual-luciferase assay using a 3'-UTR reporter containing four canonical NONO binding sites (f), RIP and real-time qRT-PCR (g) assays showing the activity and enrichment of NONO on 3'-UTR of hnRNPU , GALNT2 , and GALNT6 in MKN-45, AGS, and HeLa cells stably transfected with mock or circ-hnRNPU , and those co-transfected with NLS-mutant (mNLS) form of NONO ( n = 5) . h , Real-time qRT-PCR (normalized to β-actin , n = 5) assays showing the mRNA stability of hnRNPU , GALNT2 , and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with mNLS form of NONO or treated with actinomycin D (5 μg/ml) as indicated. i , Western blot assay indicating the expression of hnRNPU, GALNT2, and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU , and those co-transfected with mNLS form of NONO. Student’s t test or analysis of variance compared the difference in a - h . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a - i

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Activity Assay, Luciferase, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits protein glycosylation, growth, and invasion of gastric cancer cells by repressing NONO or c-Myc activity. a and b , Western blot assay showing the levels of O- and N-glycosylation in MKN-45 and AGS cells stably transfected with empty vector (mock), circ-hnRNPU , scramble shRNA (sh-Scb), or sh- circ-hnRNPU #1, and those co-transfected with NONO , c-Myc , sh-NONO #1, or sh-c-Myc #1. c and d , Representative images (left) and quantification (right) of soft agar ( c ) and matrigel invasion ( d ) assays indicating the anchorage-independent growth and invasion of MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU , sh-Scb, or sh- circ-hnRNPU #1, and those co-transfected with NONO , c-Myc , sh-NONO #1, or sh-c-Myc #1. Student’s t test compared the difference in c and d . * P < 0.05. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a - d

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Activity Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Circ-hnRNPU inhibits gastric cancer progression by repressing NONO activity in vivo. a and b , Representative imagines ( a ), in vivo growth curve ( b , left panel), and weight at the end points ( b , right panel) of xenograft tumors formed by subcutaneous injection of AGS cells stably transfected with empty vector (mock) or NONO , and those co-transfected with circ-hnRNPU into the dorsal flanks of nude mice ( n = 5 for each group). c , Representative images (upper panel) and quantification (lower panel) of immunohistochemical staining showing the expression of Ki-67 and CD31 (arrowheads) within xenograft tumors formed by hypodermic injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 5 for each group). Scale bars: 50 μm. d and e , Western blot (d) and real-time qRT-PCR (e, normalized to β-actin ) assays indicating the levels of NONO , GALNT2 , GALNT6 , MGAT1 , and hnRNPU in xenograft tumors formed by hypodermic injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 5 for each group). f , Representative images (upper panel), H&E staining (arrowheads), and quantification of lung metastatic colonization (middle panels), as well as Kaplan–Meier curves (lower panel) of nude mice treated with tail vein injection of AGS cells stably transfected with mock or NONO , and those co-transfected with circ-hnRNPU ( n = 4 for each group). Scale bar: 100 μm. Analysis of variance or Student’s t test compared the difference in b , c , e , and f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) in b , c , e , and f

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Activity Assay, In Vivo, Injection, Stable Transfection, Transfection, Plasmid Preparation, Immunohistochemistry, Staining, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circ-hnRNPU inhibits NONO-mediated c-Myc transactivation and mRNA stabilization essential for glycosylation and cancer progression

doi: 10.1186/s13046-023-02898-5

Figure Lengend Snippet: Therapeutic effects of lentivirus-mediated circ-hnRNPU over-expression in vivo. a , Representative images (left panels), in vivo growth curve (upper middle panel), weight at the end points (upper right panel), and immunohistochemical staining of Ki-67 and CD31 (lower right panels, arrowheads) of xenograft tumors formed by subcutaneous injection of MKN-45 cells into dorsal flanks of nude mice ( n = 5 for each group) that received intravenous administration of lentiviral empty vector (LV-mock) or circ-hnRNPU (LV-circ-hnRNPU) as indicated. Scale bar: 100 μm. b , Representative images (left panel), quantification of lung metastatic colonization (middle panel), and Kaplan–Meier curves (right panel) of nude mice treated with tail vein injection of MKN-45 cells and LV-mock or circ-hnRNPU (LV-circ-hnRNPU) as indicated. Scale bar: 100 μm. c , Real-time qRT-PCR (normalized to β-actin ) assay indicating the levels of NONO , c-Myc , and target genes in normal gastric mucosa ( n = 40) and gastric cancer tissues ( n = 81). d , Kaplan–Meier curves showing the overall survival of gastric cancer cases derived from KM Plotter database ( http://kmplot.com/analysis ) with low or high expression of NONO (cutoff value = 10.31), c-Myc (cutoff value = 10.63), GALNT2 (cutoff value = 8.36), GALNT6 (cutoff value = 9.14), MGAT1 (cutoff value = 9.13), or hnRNPU (cutoff value = 5.21). e , The mechanisms underlying tumor suppressive roles of circ-hnRNPU : as a circRNA derived from hnRNPU , circ-hnRNPU directly interacts with NONO to induce its cytoplasmic retention, which restrains nuclear NONO-facilitated c-Myc transactivation or cytoplasmic NONO-enhanced mRNA stability of glycosyltransferases and hnRNPU , resulting in repression of glycosylation and cancer progression. Analysis of variance or Student’s t test compared the difference in a and b . Mann–Whitney U test compared the difference in c . Log-rank test for survival comparison in b and d . * P < 0.05, ** P < 0.01. Data are shown as mean ± s.e.m. (error bars) in a - c

Article Snippet: The GST- or Flag-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU were pulled down by using Flag or GST beads (Sigma).

Techniques: Over Expression, In Vivo, Immunohistochemistry, Staining, Injection, Plasmid Preparation, Quantitative RT-PCR, Derivative Assay, Expressing, MANN-WHITNEY, Comparison

Journal: International journal of biological macromolecules

Article Title: Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment.

doi: 10.1016/j.ijbiomac.2023.125443

Figure Lengend Snippet: Fig. 3. Identification of the protein that bind to the ABCA1 G4 and regulate transcription. (A) The result of pull-down experiment showing the specific protein band for PQS 2 which was indicated by the red arrow. The sequences of oligonucleotide probes were shown. Mut: mutated ssDNA. (B) EMSA analysis showed that re combinant NONO was selectively bound to the G4 structure formed by ABCA1 G4 probe. The cold probe was the unlabelled ABCA1 G4 probe. (C) NONO ChIP enrichment around the ABCA1 G4 region in THP-1 derived macrophages and G4-KO cells indicated that the NONO bind to ABCA1 G4 in the ABCA1 promoter region. Regulation of ABCA1 protein expression through NONO overexpression (OE) in THP-1 derived macrophages (D) or G4-KO cells (E). Down-side histograms showed the percentage of the promotion of ABCA1 expression by OE. (F) RNAi-mediated inhibition of NONO resulted in decreased expression of ABCA1 and disrupted the enhancing effect of the G4 ligand on ABCA1 expression in THP-1 derived macrophages. The right-side histograms depicted the percentage of ABCA1 repression achieved through RNAi inhibition of NONO. Data were expressed as the mean ± SEM (n = 3). **p < 0.01 (Student's t-test).

Article Snippet: Recombinant NONO protein was purchased from OriGene Technologies (China).

Techniques: Derivative Assay, Expressing, Over Expression, Inhibition

Journal: International journal of biological macromolecules

Article Title: Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment.

doi: 10.1016/j.ijbiomac.2023.125443

Figure Lengend Snippet: Fig. 4. The G4 ligand promotes the binding between NONO and the ABCA1 G4. (A) Analysis of the effect of different TMPyP4 doses on the binding of the re combinant NONO to the ABCA1 G4 probe. Right-side histograms showed the percentage of promotion of TMPyP4 on the binding of NONO to the ABCA1 G4 probe. EMSA analysis of the binding affinities of NONO to single strand DNA (B) ABCA1 G4 (C) and ABCA1 G4 treated with TMPyP4 (D). (E) Binding curves of NONO to different DNAs. The Kd values were obtained by fitting the mobility shift data to the equation given in the Methods. (F) ChIP-qPCR results showed the increasing enrichment of NONO at the promoter region of ABCA1 in THP-1 derived macrophages, induced by different TMPyP4 doses (2 μM, TL; 4 μM, TH).

Article Snippet: Recombinant NONO protein was purchased from OriGene Technologies (China).

Techniques: Binding Assay, Mobility Shift, ChIP-qPCR, Derivative Assay

Journal: International journal of biological macromolecules

Article Title: Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment.

doi: 10.1016/j.ijbiomac.2023.125443

Figure Lengend Snippet: Fig. 6. The G4 ligand upregulates ABCA1 expression in primary macrophage. (A)Western blot analysis of ABCA1 protein expression in primary macrophages treated with TMPyP4. (B) RT-qPCR analysis of ABCA1 mRNA expression in primary macrophages. (C) Oil red O staining images of primary macrophages treated with TMPyP4 at different concentrations. Data were expressed as the mean ± SEM (n = 3). **p < 0.01 (Student's t-test). (D) Schematic overview of the mechanism by which NONO, ABCA1 G4 and the G4 ligand regulate ABCA1 transcription.

Article Snippet: Recombinant NONO protein was purchased from OriGene Technologies (China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Staining

Journal: OncoImmunology

Article Title: Gankyrin-mediated interaction between cancer cells and tumor-associated macrophages facilitates prostate cancer progression and androgen deprivation therapy resistance

doi: 10.1080/2162402x.2023.2173422

Figure Lengend Snippet: Figure 4. Gankyrin activates the NONO-AR-HMGB1 signaling pathway responsible for prostate cancer progression. (a), Gankyrin-interacting proteins validated by Nano LC–ESI–MS/MS and the protein–protein interaction network (STRING database) are presented. (b), Western blot analysis was utilized to identify endogenous gankyrin Co-immunoprecipitated (Co-IP) with NONO in C4-2B. IgG was detected as a control for Co-IP. (c), NONO mRNA expression was detected by RT‒PCR in gankyrin- knockdown (shGankyrin, left) or gankyrin overexpression (GankyrinOE, right) C4-2B cells and control cells. (d), Western blot analysis was utilized to identify endogenous NONO Co-immunoprecipitated with HMGB1 in C4-2B. (e), Real-time PCR assays of HMGB1 expression in gankyrin-overexpressing prostate cancer cells with or without NONO knockdown. (f), RT-PCR assays of HMGB1 expression in gankyrin knockdown prostate cancer cells without or with NONO overexpression. (g), AR expression was detected by real-time PCR in NONO-knockdown (shNONO, left) and NONO-overexpressing (NONOOE, right) C4-2B cells. (h), HMGB1 expression was detected by real-time PCR in control cells, gankyrin-overexpressing C4-2B without or with NONO or AR knockdown. (i), Quantification of the HMGB1 protein was performed in the culture media of control cells, gankyrin-overexpressing C4-2B without or with NONO or AR knockdown. (j), ChIP assay was taken to determine binding of AR to the HMGB1 promoter in control cells and gankyrin-overexpressing C4-2B. (k), Binding of AR to the HMGB1 promoter was confirmed by luciferase analyses in control cells and gankyrin-overexpressing C4-2B. AR binding was hindered in reporter gene with mutated variants (**p < .01 and ***p < .001).

Article Snippet: To analyze gankyrin, HMGB1 and NONO protein interactions, co-immunoprecipitation assays were used with antibodies against the following: gankyrin (ab182576, Abcam), HMGB1 (ab18256, Abcam), and NONO (11058, Proteintech).

Techniques: Tandem Mass Spectroscopy, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Expressing, Knockdown, Over Expression, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Luciferase

Journal: OncoImmunology

Article Title: Gankyrin-mediated interaction between cancer cells and tumor-associated macrophages facilitates prostate cancer progression and androgen deprivation therapy resistance

doi: 10.1080/2162402x.2023.2173422

Figure Lengend Snippet: Figure 5. Gankyrin facilitates prostate cancer progression via interaction between tumor cells and tumor-associated macrophages (TAMs). (a), Immunohistochemistry and H&E images of gankyrin and CD68 expression in prostate carcinoma samples (scale bar = 50 μm). (b), Spearman Correlation analysis of gankyrin and CD68 in prostate carcinoma samples. (c-d), Kaplan–Meier curves of BCR and DFS in prostate carcinoma patients in the training cohort were analyzed. (e), Pictures and counts of migrated U937 cells cultured in conditioned medium of cocultured C4-2B cells or gankyrin-overexpressing (GankyrinOE) C4-2B without or with NONO knockdown or treated with an HMGB1-neutralizing antibody (scale bar = 100 μm). (f-g), Real-time PCR was performed to analyze the expression of M1 (f) or M2 (g)-related genes in U937 cells cocultured with control C4-2B and gankyrin-overexpressing C4-2B with or without NONO knockdown or treated with an HMGB1-neutralizing antibody. (h), Representative micrographs of the invasion and migration abilities of C4-2B cultured alone or with U937 cells, with or without gankyrin overexpression in the absence or exist of NONO knockdown or treated with an HMGB1-neutralizing antibody (scale bar = 100 μm). (i), Cell viability analysis was performed in naïve C4-2B cells, enzalutamide-treated C4-2B, and enzalutamide-treated gankyrin-overexpressing C4-2B cultured alone or with U937 cells in the absence or exist of NONO knockdown or treated with an HMGB1-neutralizing antibody (*p < .05, **p < .01 and ***p < .001).

Article Snippet: To analyze gankyrin, HMGB1 and NONO protein interactions, co-immunoprecipitation assays were used with antibodies against the following: gankyrin (ab182576, Abcam), HMGB1 (ab18256, Abcam), and NONO (11058, Proteintech).

Techniques: Immunohistochemistry, Expressing, Cell Culture, Knockdown, Real-time Polymerase Chain Reaction, Control, Migration, Over Expression

Journal: OncoImmunology

Article Title: Gankyrin-mediated interaction between cancer cells and tumor-associated macrophages facilitates prostate cancer progression and androgen deprivation therapy resistance

doi: 10.1080/2162402x.2023.2173422

Figure Lengend Snippet: Figure 6. Blocking the positive regulatory loop gankyrin/NONO/HMGB1/IL-6/STAT3 inhibits ADT resistance in prostate cancer. (a), The RayBio Human Cytokine Antibody Array was performed to analyze the Cytokine profiles. Heatmap revealing the differentially expressed cytokines in conditioned medium of U937 cells cultured alone or with C4-2B cells. (b), Heatmap revealing the differentially expressed cytokines in CM U937 cells cocultured with C4-2B cells alone or with HMGB1 Ab. (c), A Venn plot of differentially expressed cytokines is presented. (d), Quantification of IL-6 protein secretion in culture media of U937 or cocultured with C4-2B in the absence or presence of HMGB1. (e), IL-6 expression was detected via RT‒PCR in U937 cells or cocultured with C4-2B in the absence or presence of HMGB1 ab. (f), Western blotting assay was utilized to evaluate the expression of STAT3 and p-STAT3 (Y705) in C4-2B or cocultured with U937 cells in the absence or presence of HMGB1ab and rIL-6. (g), Gankyrin expression was evaluated via real-time PCR in C4-2B or cocultured with U937 cells in the absence or presence of IL-6 ab or shSTAT3. (h), ChIP assay was performed to detect the binding of STAT3 to the gankyrin promoter in C4-2B cocultured with U937 cells. (i), Binding of STAT3 to the gankyrin promoter was confirmed by luciferase analyses in C4-2B cultured alone or with U937 cells. STAT3-binding sites were hindered in reporter gene with mutated version. (j), Representative H&E and immunohistochemistry micrographs for gankyrin, HMGB1, CD68, NONO, STAT3, and p-STAT3 expression in pre-ADT and post-ADT prostate cancer tissues (scale bar = 50 μm). (k-l), C4-2B or cocultured with U937 in the absence or presence of enzalutamide (ENZ), HMGB1 inhibitor glycyrrhizin (Gly) or gankyrin-targeting shRNA

Article Snippet: To analyze gankyrin, HMGB1 and NONO protein interactions, co-immunoprecipitation assays were used with antibodies against the following: gankyrin (ab182576, Abcam), HMGB1 (ab18256, Abcam), and NONO (11058, Proteintech).

Techniques: Blocking Assay, Ab Array, Cell Culture, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Immunohistochemistry, shRNA