Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: Gary boxes indicate the selected zoom-in areas. A Co-IF of NR2F1 (green) either with HP1 (red) corresponding to inactive chromatin or H3K27ac (red) corresponding to active chromatin status. B Co-IF of NR2F1 (green) with ZSCAN1 (red) and their co-localization in nucleoli (gray arrowheads). C As indicated by gray arrowheads, co-IF of NR2F2 (green) with ZSCAN1 (red) showed no nucleolar-like localization. Nucleoplasm (blue) was stained with DAPI. Scale bars: 20 µm.

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Staining

Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: Gray boxes show the selected zoom-in areas. A IF of NR2F1 by Ab H8132 (green) or by Ab #6364 (red) was performed in HeLa cells, hiPSC-derived hNCC, and undifferentiated hiPSC. Nucleolar-like foci stained by Ab H8132 are indicated by arrowheads. Ab #6364 stains endogenous NR2F1 only in the nucleoplasm and specifically in HeLa and hNCC, but not in hiPSC, which do not express NR2F1. DAPI marks necleoplasm (blue). B IF of NR2F1 Ab H8132 (green), Hoechst (blue), and two different nucleolar markers, Fibrillarin (red) and NPM1 (red). C Live-cell imaging of hiPSC NR2F1-GFP 24 h after transfection shows nuclear but not nucleolar NR2F1 localization. Zoom-in is marked with a white box and shown for the GFP channel below. Arrowheads point to NR2F1-GFP-negative nucleoli. Scale bars: 50 µm.

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Derivative Assay, Staining, Live Cell Imaging, Transfection

Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: A and B Gray boxes in the first column show the selected zoom-in area placed next to it. Nucleoplasm (blue) was stained with Hoechst. Scale bars: 50 µm. A Negative control: untransfected HEK293 cells that do not express NR2F1 endogenously. Arrowheads indicate nucleolar-like foci exclusively in the Ab3 staining. B HEK293 NR2F1 cells 48 h after transfection with an NR2F1 plasmid. Arrowheads indicate examples of nucleoli typically not stained by Ab1 and Ab2 and nucleolar-like foci stained by Ab3, seen only in cells that were unlikely to have been successfully transfected. C MFI of histograms resulting from NR2F1 staining measured for each Ab used (x-axis) in untransfected control HEK293 cells (gray columns) and in HEK293 NR2F1 cells (pink columns). The data are shown in arbitrary units (AU) and represented as mean ± SEM; the MFI was measured in at least 330 and up to 800 cells per sample. D SNRs between the MFI of HEK293 NR2F1 and untransfected HEK293 cells are shown in AU as mean ± SEM for each Ab.

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Staining, Negative Control, Transfection, Plasmid Preparation, Control

Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: A and B Gray boxes show the selected zoom-in area placed next to it. ( A - C ) The neuronal marker TUJ1 (green) was used only for IF in differentiated cells. Nucleoplasm (blue) was stained with Hoechst. Scale bars: 50 µm. The corresponding zoom-in is next to each image. A Undifferentiated WT hiPSC: arrowheads point to unspecific IF signals in the cytoplasm after Ab1 and Ab2 staining and to nucleolar-like foci after Ab3 staining. B NP/N derived from WT hiPSC. C Undifferentiated hiPSC −/− with arrowheads indicating nucleolar-like foci stained by Ab3 as well as NP/N −/− , which showed no foci. D MFI of each NR2F1 staining (Abs1–7) in AU (mean ± SEM) measured in the nucleus of undifferentiated hiPSC (gray columns) and of hiPSC-derived NP/N (magenta columns) in at least 120 cells per sample (up to 500 cells per sample). E SNRs measured for each Ab (Abs1–7) in undifferentiated WT hiPSC and hiPSC-derived WT NP/N shown in AU as mean ± SEM. F MFI measured at least in 60 and up to 150 cells per sample of each NR2F1 staining (Ab1–Ab7) displayed as mean ± SEM in undifferentiated hiPSC −/− (blue columns) and NP/N −/− (green columns).

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Marker, Staining, Derivative Assay

Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: A – F Sagittal sections of whole WT and KO mouse brains. Nucleoplasm (blue) was stained with Hoechst. A – D The overview of IF staining with Ab1 and Ab3 in WT and KO shows a strong background signal in the case of Ab3 in KO with a schematic of the anterior–posterior and dorsoventral axes shown in ( A ). Scale bars: 500 µm. E Posterior images of Nr2f1 KO . Greay boxes indicate the area of zoom-in shown next to it. F Anterior areas and posterior areas in WT . Gray boxes in E and F indicate zoom-in areas. Only Abs1-4 can detect Nr2f1, but Ab3 leads to high background staining localized in the cytoplasm or extracellular matrix rather than in the nucleoli. Scale bars: 50 µm in the first column of E and first and second columns of F ; 25 µm in the last column of ( E ) and ( F ). G MFI of each Nr2f1 staining measured in random fields from at least 6 and up to 12 different cortical regions per sample in Nr2f1 KO and WT brains represented as mean ± SEM in AU. H SNRs ( WT vs. KO ).

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Staining

Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: Gray boxes show the selected zoom-in area placed next to it; arrowheads indicate nucleolar areas. A – D Co-staining of NR2F1 with Ab1 (green) and Ab3 (red) in untransfected HEK293 cells and 48 h post-transfection in HEK293 NR2F1 cells. Nucleoplasmi was stained by Hoechst. Only successfully transfected HEK293 NR2F1 cells display double-positive nuclei co-stained by Ab1 and Ab3. A PFA fixation for 15 min and B 2 h reduced the unspecific nucleolar-like foci resulting from Ab3 staining without affecting the Ab1 signal. C 15 min and D 2 h fixation with 70% ethanol completely prevented the occurrence of unspecific nucleolar-like foci in the Ab3 staining without affecting the Ab1 staining. Scale bars: 50 µm. E and F Scale bars: 59 µm. Co-staining of fibrillarin (red) with Ab1, Ab2, and Ab3 (red). E – F Nucleoplasm was stained by DAPI (blue). E WT hPSC shows a specific nucleolar pattern stained by Ab3 that overlaps with fibrillarin in DAPI-depleted regions. F In WT hNCC cells, all Abs stain the NR2F1 nucleus (DAPI-positive but fibrillarin-negative regions), but only Ab3 overlaps with fibrillarin in DAPI-depleted regions.

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Staining, Transfection

Journal: Communications Biology

Article Title: Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

doi: 10.1038/s42003-025-07985-1

Figure Lengend Snippet: A – D Quantification of NR2F1 levels by FC using all seven Abs in HEK293 and HEK293 NR2F1 cells. A and B Untransfected and C and D HEK293 NR2F1 cells were fixed with 70% ethanol 48 h after transfection, and stained with the primary Abs, followed by adequate AF488 as a secondary Ab. A and C FC data. For each sample, two batches with each 10,000 events were measured using the same settings and show that Ab1, Ab3, and Ab4 detect NR2F1 best under these conditions. B and D After FC measurement, the cell suspensions of the remaining samples were stained with Hoechst (blue), spotted on glass slides, and visualized by fluorescence microscopy. Scale bars: 50 µm. E and F Detection of NR2F1 in WB in samples from WT hiPSC and hNCC as well as hNCC −/− and hNCC +/− . 20 µg per sample of RIPA lysate was loaded. Red arrows mark the 44–46 kDa band corresponding to the specific size of NR2F1, blue arrows mark GAPDH (~36 kDa) used as loading control. E Undifferentiated WT hiPSC and hNCC. F hNCC −/− and hNCC +/− . All Abs recognized the 44–46 kDa band corresponding to NR2F1 hNCC +/− , and, depending on the Ab, several other bands. Ab6 and Ab7 also recognized the 44–46 kDa band in hNCC −/− , which was not detected by other Abs. M molecular size marker.

Article Snippet: In addition to Ab H8132, we also used another anti-NR2F1 Ab (#6364, Cell Signaling), which targets a different region of NR2F1.

Techniques: Transfection, Staining, Fluorescence, Microscopy, Control, Marker

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 1 Screening of NR2F1 in high-plasticity mouse models and ENZ-R cells. (A, B) Schematic representation of the establishment of the high-plasticity mouse model and ENZ-R PCa cellular model. (C–E) Volcano plots illustrating the DEGs between 16DCRPC and 42DENZR; LNCaPCON and LNCaPENZR; and C4-2CON and C4-2ENZR. Red dots represent upregulated genes, while green dots represent downregulated genes. (F) PCA depicting the distribution of transcriptional sequencing data across six cells. (G) Venn diagram displaying the overlapping genes among the three groups. (H, I) Validation of the RNA and protein expression of NR2F1 in C4-2ENZR and C4-2BENZR cells, with C4-2CON and C4-2BCON cells serving as controls, through RT-qPCR and WB assays. (J, K) RT-qPCR and WB assays validated the expression of NR2F1 in PCa cell lines compared to a normal prostate epithelial cell line (RWPE1). **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The membranes were then blocked with TBS containing 5% nonfat milk and co-incubated overnight at 4 °C with specific antibodies: anti-rabbit β-actin (#4970, CST, 1:1000); antirabbit NR2F1 (#6364, CST); antibodies from the Cell Cycle Regulation Antibody Sampler Kit (#9932, CST, including CDK2, CDK4, CDK6, p21, p27, cyclin D1); anti-rabbit SNAIL (#9585, CST); anti-β-catenin (#8480, CST); rabbit anti-E-cadherin (#3195, CST); anti-mouse PHB2 (66424-1-Ig, Proteintech, China); anti-rabbit ESR1 (21244-1-AP, Proteintech); anti-rabbit androgen receptor (AR) (22089-1-AP, Proteintech); anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech); anti-rabbit SRSF1 (12929-2-AP, Proteintech); anti-rabbit CD63 (25682-1- AP, Proteintech); anti-rabbit HSP70 (10995-1-AP, Proteintech); anti-rabbit lamin-A (10298-1-AP, Proteintech); anti-rabbit ERK 1/2 (T40071, Abmart); anti-rabbit phospho-ERK 1/2 (T40072, Abmart); anti-rabbit p38 MAPK (T55600, Abmart); and anti-rabbit phospho-p38 MAPK (TA4001, Abmart).

Techniques: Sequencing, Biomarker Discovery, Expressing, Quantitative RT-PCR

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 3 Transcriptional sequencing analysis of NR2F1 depletion in ENZ-R and control cells. (A, B) Heat map demonstrating the clustering of DEGs. (C) DEGs in C4-2 and C4-2ENZR cells after NR2F1 silencing, with log2 (fold change) > 0.5 and p-value < 0.05. (D–I) GO analysis of CCs, MFs, and BPs for DEGs in C4-2 and C4-2ENZR cells after NR2F1 silencing. (J, K) The results of the KEGG analysis are presented. (L) Venn diagram illustrating the intersection of DEGs in C4-2 and C4-2ENZR cells after NR2F1 silencing. (M–O) CC, BP, and KEGG analyses were performed on the intersecting DEGs

Article Snippet: The membranes were then blocked with TBS containing 5% nonfat milk and co-incubated overnight at 4 °C with specific antibodies: anti-rabbit β-actin (#4970, CST, 1:1000); antirabbit NR2F1 (#6364, CST); antibodies from the Cell Cycle Regulation Antibody Sampler Kit (#9932, CST, including CDK2, CDK4, CDK6, p21, p27, cyclin D1); anti-rabbit SNAIL (#9585, CST); anti-β-catenin (#8480, CST); rabbit anti-E-cadherin (#3195, CST); anti-mouse PHB2 (66424-1-Ig, Proteintech, China); anti-rabbit ESR1 (21244-1-AP, Proteintech); anti-rabbit androgen receptor (AR) (22089-1-AP, Proteintech); anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech); anti-rabbit SRSF1 (12929-2-AP, Proteintech); anti-rabbit CD63 (25682-1- AP, Proteintech); anti-rabbit HSP70 (10995-1-AP, Proteintech); anti-rabbit lamin-A (10298-1-AP, Proteintech); anti-rabbit ERK 1/2 (T40071, Abmart); anti-rabbit phospho-ERK 1/2 (T40072, Abmart); anti-rabbit p38 MAPK (T55600, Abmart); and anti-rabbit phospho-p38 MAPK (TA4001, Abmart).

Techniques: Sequencing, Control

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 4 Functional enrichment analysis of DEGs in patients with PCa exhibiting high or low NR2F1 expression. (A) PCA illustrates the clustering of RNA-seq data from patients with PCa exhibiting high or low NR2F1 expression. (B) A heat map presents the top 50 DEGs across various clinical patient groups. (C) The top four pathways identified through different GO analyses are depicted. (D) The top 10 enriched pathways from the KEGG analysis are displayed. (E) Pathways with NES scores > 2 in the GSEA of DEGs are shown

Article Snippet: The membranes were then blocked with TBS containing 5% nonfat milk and co-incubated overnight at 4 °C with specific antibodies: anti-rabbit β-actin (#4970, CST, 1:1000); antirabbit NR2F1 (#6364, CST); antibodies from the Cell Cycle Regulation Antibody Sampler Kit (#9932, CST, including CDK2, CDK4, CDK6, p21, p27, cyclin D1); anti-rabbit SNAIL (#9585, CST); anti-β-catenin (#8480, CST); rabbit anti-E-cadherin (#3195, CST); anti-mouse PHB2 (66424-1-Ig, Proteintech, China); anti-rabbit ESR1 (21244-1-AP, Proteintech); anti-rabbit androgen receptor (AR) (22089-1-AP, Proteintech); anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech); anti-rabbit SRSF1 (12929-2-AP, Proteintech); anti-rabbit CD63 (25682-1- AP, Proteintech); anti-rabbit HSP70 (10995-1-AP, Proteintech); anti-rabbit lamin-A (10298-1-AP, Proteintech); anti-rabbit ERK 1/2 (T40071, Abmart); anti-rabbit phospho-ERK 1/2 (T40072, Abmart); anti-rabbit p38 MAPK (T55600, Abmart); and anti-rabbit phospho-p38 MAPK (TA4001, Abmart).

Techniques: Functional Assay, Expressing, RNA Sequencing

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 5 NR2F1-AS1 upregulates the expression of NR2F1 via binding to SRSF1. (A) Strong correlation between NR2F1 and NR2F1-AS1 in TCGA-PCa cohort. (B) RT-qPCR analysis of NR2F1-AS1 expression following NR2F1 overexpression or knockdown in C4-2ENZR cells. (C) RT-qPCR analysis of the mRNA expres sion of NR2F1 subsequent to NR2F1-AS1 overexpression in C4-2ENZR and PC-3 cells. (D) NR2F1 expression levels in control and NR2F1-AS1 pulldown assays. (E) Silver staining assays conducted on protein products obtained from NR2F1-AS1 pulldown assays, with poly(A)25 as a control. (F) Most detected RNA-binding proteins were predicted using the CATRAPID website. (G) Validation of SRSF1 expression through WB in NR2F1-AS1 pulldown assays. (H-J) RIP assays were conducted using SRSF1 antibodies in C4-2ENZR cells. Protein levels of SRSF1 and RNA levels of NR2F1 or NR2F1-AS1 were detected in RIP and IgG groups. (K) Schematic representation of plasmid construction for wild type and mutated NR2F1-AS1. (L) Prediction of binding sites between NR2F1-AS1 and SRSF1 using CATRAPID. (M) RNA levels of NR2F1-AS1 and NR2F1 assessed through PCR and agarose-gel electrophoresis using products of RIP assays following overexpression of wild type and mutated NR2F1-AS1. (N) RT-qPCR analysis of SRSF1, NR2F1, and NR2F1-AS1 RNA levels after SRSF1 depletion in C4-2ENZR cells. **p < 0.001, ***p < 0.0001, ****p < 0.0001

Article Snippet: The membranes were then blocked with TBS containing 5% nonfat milk and co-incubated overnight at 4 °C with specific antibodies: anti-rabbit β-actin (#4970, CST, 1:1000); antirabbit NR2F1 (#6364, CST); antibodies from the Cell Cycle Regulation Antibody Sampler Kit (#9932, CST, including CDK2, CDK4, CDK6, p21, p27, cyclin D1); anti-rabbit SNAIL (#9585, CST); anti-β-catenin (#8480, CST); rabbit anti-E-cadherin (#3195, CST); anti-mouse PHB2 (66424-1-Ig, Proteintech, China); anti-rabbit ESR1 (21244-1-AP, Proteintech); anti-rabbit androgen receptor (AR) (22089-1-AP, Proteintech); anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech); anti-rabbit SRSF1 (12929-2-AP, Proteintech); anti-rabbit CD63 (25682-1- AP, Proteintech); anti-rabbit HSP70 (10995-1-AP, Proteintech); anti-rabbit lamin-A (10298-1-AP, Proteintech); anti-rabbit ERK 1/2 (T40071, Abmart); anti-rabbit phospho-ERK 1/2 (T40072, Abmart); anti-rabbit p38 MAPK (T55600, Abmart); and anti-rabbit phospho-p38 MAPK (TA4001, Abmart).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Over Expression, Knockdown, Control, Silver Staining, RNA Binding Assay, Biomarker Discovery, Plasmid Preparation, Agarose Gel Electrophoresis

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 6 NR2F1 stabilizes hormonal receptor complexes to sustain ESR1 and AR expression. (A) Silver staining elucidated the differential protein bands of NR2F1 in Co-IP assays, with red arrows indicating the bands. (B, C) The top 10 identified proteins from two distinct bands through LC-MS analysis. (D) Venn diagram illustrating the overlap of DEGs after NR2F1 silencing in C4-2ENZR cells and proteins of two bands identified through LC-MS analysis. (E) Prediction of potential interacting proteins using the STRING website. (F, G) Validation of protein expression levels of NR2F1, PHB2, and ESR1 via WB following Co-IP assays of NR2F1 and PHB2 in C4-2ENZR and C4-2BENZR cells. (H) Related scores of the binding possibilities of ESR1-NR2F1, NR2F1-PHB2, and ESR1-PHB2 inter actions, as predicted using Alpha-Fold Multimer. (I) Prediction of binding sites and protein secondary structure simulation via Alpha-Fold Multimer. (J) IF experiments validated the colocalization of NR2F1-PHB2, PHB2-AR, and ESR1-PHB2 following NR2F1 depletion in C4-2ENZR cells. (K, L) Enriched pathways were identified through GSEA of DEGs in C4-2ENZR cells after NR2F1 silencing. (M) Measurement of PHB2, ESR1, and AR expression levels following NR2F1 knockdown or overexpression in C4-2ENZR and C4-2BENZR cells via WB assays

Article Snippet: The membranes were then blocked with TBS containing 5% nonfat milk and co-incubated overnight at 4 °C with specific antibodies: anti-rabbit β-actin (#4970, CST, 1:1000); antirabbit NR2F1 (#6364, CST); antibodies from the Cell Cycle Regulation Antibody Sampler Kit (#9932, CST, including CDK2, CDK4, CDK6, p21, p27, cyclin D1); anti-rabbit SNAIL (#9585, CST); anti-β-catenin (#8480, CST); rabbit anti-E-cadherin (#3195, CST); anti-mouse PHB2 (66424-1-Ig, Proteintech, China); anti-rabbit ESR1 (21244-1-AP, Proteintech); anti-rabbit androgen receptor (AR) (22089-1-AP, Proteintech); anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech); anti-rabbit SRSF1 (12929-2-AP, Proteintech); anti-rabbit CD63 (25682-1- AP, Proteintech); anti-rabbit HSP70 (10995-1-AP, Proteintech); anti-rabbit lamin-A (10298-1-AP, Proteintech); anti-rabbit ERK 1/2 (T40071, Abmart); anti-rabbit phospho-ERK 1/2 (T40072, Abmart); anti-rabbit p38 MAPK (T55600, Abmart); and anti-rabbit phospho-p38 MAPK (TA4001, Abmart).

Techniques: Expressing, Silver Staining, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Biomarker Discovery, Binding Assay, Knockdown, Over Expression

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 7 Exosomal NR2F1 and NR2F1-AS1 facilitate the proliferation of ENZ-R cells via HnRNPA2B1 binding. (A) Venn diagram illustrates gene overlap be tween NR2F1 Co-IP assays and NR2F1-AS1 pulldown assays. (B, C) RNA pulldown and RIP assays validated mutual binding of NR2F1-AS1 and HnRNPA2B1. (D) Co-IP assays validated the mutual binding between NR2F1 and HnRNPA2B1. (E) The binding sites and protein structures were predicted and simulated using Alpha-Fold Multimer. (F) Colocalization of NR2F1 and HnRNPA2B1 detected by IF in both C4-2 and C4-2ENZR cells. (G) Electron microscope images captured the morphology of exosomes from NR2F1-overexpressing and control C4-2ENZR cells. (H) NTA revealed the size and concentration of isolated exosomes. (I) WB analysis indicated the levels of CD63, HSP70, lamin-A, and NR2F1 in exosomes from different groups. (J) PCR and agarose-gel electro phoresis elucidated NR2F1-AS1 expression in exosomes from NR2F1 overexpression and control groups. (K–M) CCK-8 and EdU assays demonstrated the proliferation abilities of C4-2ENZR cells treated with exosomes extracted from NR2F1-overexpressing and control cells, with PBS as the Control group. Exos referred to exosome. Oe-NR referred to overexpression of NR2F1. (N, O) IF experiments showed the colocalization of NR2F1 and HnRNPA2B1 in C4-2ENZR cells after treatment with exosomes from NR2F1-overexpressing and control cells. *p < 0.05, **p < 0.01, ****p < 0.0001

Article Snippet: The membranes were then blocked with TBS containing 5% nonfat milk and co-incubated overnight at 4 °C with specific antibodies: anti-rabbit β-actin (#4970, CST, 1:1000); antirabbit NR2F1 (#6364, CST); antibodies from the Cell Cycle Regulation Antibody Sampler Kit (#9932, CST, including CDK2, CDK4, CDK6, p21, p27, cyclin D1); anti-rabbit SNAIL (#9585, CST); anti-β-catenin (#8480, CST); rabbit anti-E-cadherin (#3195, CST); anti-mouse PHB2 (66424-1-Ig, Proteintech, China); anti-rabbit ESR1 (21244-1-AP, Proteintech); anti-rabbit androgen receptor (AR) (22089-1-AP, Proteintech); anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech); anti-rabbit SRSF1 (12929-2-AP, Proteintech); anti-rabbit CD63 (25682-1- AP, Proteintech); anti-rabbit HSP70 (10995-1-AP, Proteintech); anti-rabbit lamin-A (10298-1-AP, Proteintech); anti-rabbit ERK 1/2 (T40071, Abmart); anti-rabbit phospho-ERK 1/2 (T40072, Abmart); anti-rabbit p38 MAPK (T55600, Abmart); and anti-rabbit phospho-p38 MAPK (TA4001, Abmart).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Microscopy, Control, Concentration Assay, Isolation, Agarose Gel Electrophoresis, Expressing, Over Expression, CCK-8 Assay

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 1 Screening of NR2F1 in high-plasticity mouse models and ENZ-R cells. (A, B) Schematic representation of the establishment of the high-plasticity mouse model and ENZ-R PCa cellular model. (C–E) Volcano plots illustrating the DEGs between 16DCRPC and 42DENZR; LNCaPCON and LNCaPENZR; and C4-2CON and C4-2ENZR. Red dots represent upregulated genes, while green dots represent downregulated genes. (F) PCA depicting the distribution of transcriptional sequencing data across six cells. (G) Venn diagram displaying the overlapping genes among the three groups. (H, I) Validation of the RNA and protein expression of NR2F1 in C4-2ENZR and C4-2BENZR cells, with C4-2CON and C4-2BCON cells serving as controls, through RT-qPCR and WB assays. (J, K) RT-qPCR and WB assays validated the expression of NR2F1 in PCa cell lines compared to a normal prostate epithelial cell line (RWPE1). **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Cell protein supernatants in lysis buffer were initially processed with 50 μL of A/G beads (Merck, USA), followed by overnight incubation at 4 °C with 5 μg of either control IgG (30000-0-AP, Proteintech) or anti-rabbit NR2F1 (#6364, CST, 1:100), anti-rabbit PHB2 (12295- 1-AP, Proteintech, 1:100), and anti-rabbit HnRNPA2B1 (ab31645, Abcam, 1:100) antibodies.

Techniques: Sequencing, Biomarker Discovery, Expressing, Quantitative RT-PCR

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 3 Transcriptional sequencing analysis of NR2F1 depletion in ENZ-R and control cells. (A, B) Heat map demonstrating the clustering of DEGs. (C) DEGs in C4-2 and C4-2ENZR cells after NR2F1 silencing, with log2 (fold change) > 0.5 and p-value < 0.05. (D–I) GO analysis of CCs, MFs, and BPs for DEGs in C4-2 and C4-2ENZR cells after NR2F1 silencing. (J, K) The results of the KEGG analysis are presented. (L) Venn diagram illustrating the intersection of DEGs in C4-2 and C4-2ENZR cells after NR2F1 silencing. (M–O) CC, BP, and KEGG analyses were performed on the intersecting DEGs

Article Snippet: Cell protein supernatants in lysis buffer were initially processed with 50 μL of A/G beads (Merck, USA), followed by overnight incubation at 4 °C with 5 μg of either control IgG (30000-0-AP, Proteintech) or anti-rabbit NR2F1 (#6364, CST, 1:100), anti-rabbit PHB2 (12295- 1-AP, Proteintech, 1:100), and anti-rabbit HnRNPA2B1 (ab31645, Abcam, 1:100) antibodies.

Techniques: Sequencing, Control

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 4 Functional enrichment analysis of DEGs in patients with PCa exhibiting high or low NR2F1 expression. (A) PCA illustrates the clustering of RNA-seq data from patients with PCa exhibiting high or low NR2F1 expression. (B) A heat map presents the top 50 DEGs across various clinical patient groups. (C) The top four pathways identified through different GO analyses are depicted. (D) The top 10 enriched pathways from the KEGG analysis are displayed. (E) Pathways with NES scores > 2 in the GSEA of DEGs are shown

Article Snippet: Cell protein supernatants in lysis buffer were initially processed with 50 μL of A/G beads (Merck, USA), followed by overnight incubation at 4 °C with 5 μg of either control IgG (30000-0-AP, Proteintech) or anti-rabbit NR2F1 (#6364, CST, 1:100), anti-rabbit PHB2 (12295- 1-AP, Proteintech, 1:100), and anti-rabbit HnRNPA2B1 (ab31645, Abcam, 1:100) antibodies.

Techniques: Functional Assay, Expressing, RNA Sequencing

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 5 NR2F1-AS1 upregulates the expression of NR2F1 via binding to SRSF1. (A) Strong correlation between NR2F1 and NR2F1-AS1 in TCGA-PCa cohort. (B) RT-qPCR analysis of NR2F1-AS1 expression following NR2F1 overexpression or knockdown in C4-2ENZR cells. (C) RT-qPCR analysis of the mRNA expres sion of NR2F1 subsequent to NR2F1-AS1 overexpression in C4-2ENZR and PC-3 cells. (D) NR2F1 expression levels in control and NR2F1-AS1 pulldown assays. (E) Silver staining assays conducted on protein products obtained from NR2F1-AS1 pulldown assays, with poly(A)25 as a control. (F) Most detected RNA-binding proteins were predicted using the CATRAPID website. (G) Validation of SRSF1 expression through WB in NR2F1-AS1 pulldown assays. (H-J) RIP assays were conducted using SRSF1 antibodies in C4-2ENZR cells. Protein levels of SRSF1 and RNA levels of NR2F1 or NR2F1-AS1 were detected in RIP and IgG groups. (K) Schematic representation of plasmid construction for wild type and mutated NR2F1-AS1. (L) Prediction of binding sites between NR2F1-AS1 and SRSF1 using CATRAPID. (M) RNA levels of NR2F1-AS1 and NR2F1 assessed through PCR and agarose-gel electrophoresis using products of RIP assays following overexpression of wild type and mutated NR2F1-AS1. (N) RT-qPCR analysis of SRSF1, NR2F1, and NR2F1-AS1 RNA levels after SRSF1 depletion in C4-2ENZR cells. **p < 0.001, ***p < 0.0001, ****p < 0.0001

Article Snippet: Cell protein supernatants in lysis buffer were initially processed with 50 μL of A/G beads (Merck, USA), followed by overnight incubation at 4 °C with 5 μg of either control IgG (30000-0-AP, Proteintech) or anti-rabbit NR2F1 (#6364, CST, 1:100), anti-rabbit PHB2 (12295- 1-AP, Proteintech, 1:100), and anti-rabbit HnRNPA2B1 (ab31645, Abcam, 1:100) antibodies.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Over Expression, Knockdown, Control, Silver Staining, RNA Binding Assay, Biomarker Discovery, Plasmid Preparation, Agarose Gel Electrophoresis

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 6 NR2F1 stabilizes hormonal receptor complexes to sustain ESR1 and AR expression. (A) Silver staining elucidated the differential protein bands of NR2F1 in Co-IP assays, with red arrows indicating the bands. (B, C) The top 10 identified proteins from two distinct bands through LC-MS analysis. (D) Venn diagram illustrating the overlap of DEGs after NR2F1 silencing in C4-2ENZR cells and proteins of two bands identified through LC-MS analysis. (E) Prediction of potential interacting proteins using the STRING website. (F, G) Validation of protein expression levels of NR2F1, PHB2, and ESR1 via WB following Co-IP assays of NR2F1 and PHB2 in C4-2ENZR and C4-2BENZR cells. (H) Related scores of the binding possibilities of ESR1-NR2F1, NR2F1-PHB2, and ESR1-PHB2 inter actions, as predicted using Alpha-Fold Multimer. (I) Prediction of binding sites and protein secondary structure simulation via Alpha-Fold Multimer. (J) IF experiments validated the colocalization of NR2F1-PHB2, PHB2-AR, and ESR1-PHB2 following NR2F1 depletion in C4-2ENZR cells. (K, L) Enriched pathways were identified through GSEA of DEGs in C4-2ENZR cells after NR2F1 silencing. (M) Measurement of PHB2, ESR1, and AR expression levels following NR2F1 knockdown or overexpression in C4-2ENZR and C4-2BENZR cells via WB assays

Article Snippet: Cell protein supernatants in lysis buffer were initially processed with 50 μL of A/G beads (Merck, USA), followed by overnight incubation at 4 °C with 5 μg of either control IgG (30000-0-AP, Proteintech) or anti-rabbit NR2F1 (#6364, CST, 1:100), anti-rabbit PHB2 (12295- 1-AP, Proteintech, 1:100), and anti-rabbit HnRNPA2B1 (ab31645, Abcam, 1:100) antibodies.

Techniques: Expressing, Silver Staining, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Biomarker Discovery, Binding Assay, Knockdown, Over Expression

Journal: Journal of nanobiotechnology

Article Title: Exosome-delivered NR2F1-AS1 and NR2F1 drive phenotypic transition from dormancy to proliferation in treatment-resistant prostate cancer via stabilizing hormonal receptors.

doi: 10.1186/s12951-024-03025-y

Figure Lengend Snippet: Fig. 7 Exosomal NR2F1 and NR2F1-AS1 facilitate the proliferation of ENZ-R cells via HnRNPA2B1 binding. (A) Venn diagram illustrates gene overlap be tween NR2F1 Co-IP assays and NR2F1-AS1 pulldown assays. (B, C) RNA pulldown and RIP assays validated mutual binding of NR2F1-AS1 and HnRNPA2B1. (D) Co-IP assays validated the mutual binding between NR2F1 and HnRNPA2B1. (E) The binding sites and protein structures were predicted and simulated using Alpha-Fold Multimer. (F) Colocalization of NR2F1 and HnRNPA2B1 detected by IF in both C4-2 and C4-2ENZR cells. (G) Electron microscope images captured the morphology of exosomes from NR2F1-overexpressing and control C4-2ENZR cells. (H) NTA revealed the size and concentration of isolated exosomes. (I) WB analysis indicated the levels of CD63, HSP70, lamin-A, and NR2F1 in exosomes from different groups. (J) PCR and agarose-gel electro phoresis elucidated NR2F1-AS1 expression in exosomes from NR2F1 overexpression and control groups. (K–M) CCK-8 and EdU assays demonstrated the proliferation abilities of C4-2ENZR cells treated with exosomes extracted from NR2F1-overexpressing and control cells, with PBS as the Control group. Exos referred to exosome. Oe-NR referred to overexpression of NR2F1. (N, O) IF experiments showed the colocalization of NR2F1 and HnRNPA2B1 in C4-2ENZR cells after treatment with exosomes from NR2F1-overexpressing and control cells. *p < 0.05, **p < 0.01, ****p < 0.0001

Article Snippet: Cell protein supernatants in lysis buffer were initially processed with 50 μL of A/G beads (Merck, USA), followed by overnight incubation at 4 °C with 5 μg of either control IgG (30000-0-AP, Proteintech) or anti-rabbit NR2F1 (#6364, CST, 1:100), anti-rabbit PHB2 (12295- 1-AP, Proteintech, 1:100), and anti-rabbit HnRNPA2B1 (ab31645, Abcam, 1:100) antibodies.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Microscopy, Control, Concentration Assay, Isolation, Agarose Gel Electrophoresis, Expressing, Over Expression, CCK-8 Assay