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 principal antibodies listed below were employed: anti-rabbit NR2F1 (24573-1-AP, Proteintech, 1:100), anti-mouse PHB2 (66424-1-Ig, Proteintech, 1:100), anti-rabbit ESR1 (21244-1-AP, Proteintech, 1:100), anti-rabbit AR (22089- 1-AP, Proteintech, 1:100), and anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech, 1:100).

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 principal antibodies listed below were employed: anti-rabbit NR2F1 (24573-1-AP, Proteintech, 1:100), anti-mouse PHB2 (66424-1-Ig, Proteintech, 1:100), anti-rabbit ESR1 (21244-1-AP, Proteintech, 1:100), anti-rabbit AR (22089- 1-AP, Proteintech, 1:100), and anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech, 1:100).

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 principal antibodies listed below were employed: anti-rabbit NR2F1 (24573-1-AP, Proteintech, 1:100), anti-mouse PHB2 (66424-1-Ig, Proteintech, 1:100), anti-rabbit ESR1 (21244-1-AP, Proteintech, 1:100), anti-rabbit AR (22089- 1-AP, Proteintech, 1:100), and anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech, 1:100).

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 principal antibodies listed below were employed: anti-rabbit NR2F1 (24573-1-AP, Proteintech, 1:100), anti-mouse PHB2 (66424-1-Ig, Proteintech, 1:100), anti-rabbit ESR1 (21244-1-AP, Proteintech, 1:100), anti-rabbit AR (22089- 1-AP, Proteintech, 1:100), and anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech, 1:100).

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 principal antibodies listed below were employed: anti-rabbit NR2F1 (24573-1-AP, Proteintech, 1:100), anti-mouse PHB2 (66424-1-Ig, Proteintech, 1:100), anti-rabbit ESR1 (21244-1-AP, Proteintech, 1:100), anti-rabbit AR (22089- 1-AP, Proteintech, 1:100), and anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech, 1:100).

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 principal antibodies listed below were employed: anti-rabbit NR2F1 (24573-1-AP, Proteintech, 1:100), anti-mouse PHB2 (66424-1-Ig, Proteintech, 1:100), anti-rabbit ESR1 (21244-1-AP, Proteintech, 1:100), anti-rabbit AR (22089- 1-AP, Proteintech, 1:100), and anti-mouse HnRNPA2B1 (67445-1-Ig, Proteintech, 1:100).

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