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: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A The expression levels of H3K27Ac were analyzed by Western blotting in normal cells, in SF CRC cells and in SF CRC cells treated with ETO. B The interaction of H3K27Ac and Nanog promoter in SF CRC cells was assessed by CHIP-PCR. IgG was used as negative control. Scheme of the different positions of ChIP-PCR primers was also shown (P1: −1844/−823 bp, P2: −1043/−1024 bp, P3: −791/−772 bp, P4: −259/−236 bp). C The expression levels of histone acetyltransferases ( P300, KAT2A, KAT3A ) and histone deacetylases ( HDAC1, HDAC2 ) were examined by PCR in normal HT29, in SF HT29 cells and in SF HT29 cells treated with ETO. D The expression level of P300 was analyzed by Western blotting in normal CRC cells, in serum-deprived CRC cells and in serum-deprived CRC cells treated with ETO. E , F P300 inhibition downregulated the expression of Nanog and H3K27Ac. The serum-deprived HT29 and HCT116 cells were treated with C646 for 24 h. Then, the cells were tested for Nanog expression by PCR (E) and Nanog and H3K27Ac expression by Western blotting (F) . C646: P300 inhibitor. Data are shown as the means ± s.e.m., n = 3; ns: no statistical significance, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A – C The expression of ACLY was analyzed by PCR (A) , Western blotting (B) and Immunofluorescence staining (C) in normal CRC cells, in SF CRC cells and in SF CRC cells treated with ETO. Scale bar = 20 μm. D The expression of ACLY in cytoplasm and nucleus was analyzed by Western blotting. E , F ACLY knockdown decreased the expression of P300 and Nanog. Serum deprived HT29 cells were transfected with ACLY siRNAs or negative control siRNAs. The expression of ACLY , P300 and Nanog was analyzed by PCR. F Western blotting analysis of P300, H3K27Ac and Nanog expression by ACLY inhibition in serum-deprived CRC cells. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A , B Dormancy markers could be inhibited by ACLY inhibition and reversed by Nanog overexpression in SF HT29 cells. C , D Dormancy markers could be inhibited by P300 inhibition and reversed by Nanog overexpression in SF HT29 cells. E CD31 (green) and Nanog (red) expression in subcutaneous tumors of mouse and CRC tissues by Immunofluorescence. The stained sections were observed under a fluorescent microscope and photographed. Scale bar = 50 μm. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Inhibition, Over Expression, Expressing, Immunofluorescence, Staining, Microscopy

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: FAO/ACLY signaling promoted Nanog expression via P300/H3K27Ac regulation. Then, Nanog upregulation increased the transcription of P21, P27,COUP-TF1, and hDEC2, and then induced cellular dormancy of serum deprived CRC cells.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A Nanog overexpression increased the cell viability of SF CRC cells. CRC cells were transfected with pCMV3-Nanog plasmids or empty vector for 48 h before serum deprivation. Cell viability was detected by CCK8 and marked as SF 0–96 h. B Nanog overexpression increased the percentage of CRC cells in G0-G1 phase. CRC cells were transfected with pCMV3-Nanog plasmids or empty vector for 48 h before analyzing in FCM. C , D Nanog overexpression increased the expression of COUP-TF1, hDEC2, P21 and P27. CRC cells were transfected with pCMV3-Nanog plasmids for 48 h. The empty vector pCMV3 plasmids were used as controls. The expression of COUP-TF1, hDEC2, P21, and P27 was tested by PCR and Western blotting. The relative expression was quantified by Image J. E SF HT29 cells were collected for ChIP-PCR with anti-Nanog antibody and specific primers for P21 and P27. IgG was used as negative control. Scheme of the different positions of ChIP-PCR primers was also shown (P21-p1: −1366/−1347 bp, P21-p2: −420/−399 bp, P27-p1: −977/ −958 bp, P27-p2: −698/−679 bp). Data are shown as the means ± s.e.m., n = 3; ** P < 0.01, *** P < 0.001. Data represented at least 3 independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A The expression levels of H3K27Ac were analyzed by Western blotting in normal cells, in SF CRC cells and in SF CRC cells treated with ETO. B The interaction of H3K27Ac and Nanog promoter in SF CRC cells was assessed by CHIP-PCR. IgG was used as negative control. Scheme of the different positions of ChIP-PCR primers was also shown (P1: −1844/−823 bp, P2: −1043/−1024 bp, P3: −791/−772 bp, P4: −259/−236 bp). C The expression levels of histone acetyltransferases ( P300, KAT2A, KAT3A ) and histone deacetylases ( HDAC1, HDAC2 ) were examined by PCR in normal HT29, in SF HT29 cells and in SF HT29 cells treated with ETO. D The expression level of P300 was analyzed by Western blotting in normal CRC cells, in serum-deprived CRC cells and in serum-deprived CRC cells treated with ETO. E , F P300 inhibition downregulated the expression of Nanog and H3K27Ac. The serum-deprived HT29 and HCT116 cells were treated with C646 for 24 h. Then, the cells were tested for Nanog expression by PCR (E) and Nanog and H3K27Ac expression by Western blotting (F) . C646: P300 inhibitor. Data are shown as the means ± s.e.m., n = 3; ns: no statistical significance, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A – C The expression of ACLY was analyzed by PCR (A) , Western blotting (B) and Immunofluorescence staining (C) in normal CRC cells, in SF CRC cells and in SF CRC cells treated with ETO. Scale bar = 20 μm. D The expression of ACLY in cytoplasm and nucleus was analyzed by Western blotting. E , F ACLY knockdown decreased the expression of P300 and Nanog. Serum deprived HT29 cells were transfected with ACLY siRNAs or negative control siRNAs. The expression of ACLY , P300 and Nanog was analyzed by PCR. F Western blotting analysis of P300, H3K27Ac and Nanog expression by ACLY inhibition in serum-deprived CRC cells. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A , B Dormancy markers could be inhibited by ACLY inhibition and reversed by Nanog overexpression in SF HT29 cells. C , D Dormancy markers could be inhibited by P300 inhibition and reversed by Nanog overexpression in SF HT29 cells. E CD31 (green) and Nanog (red) expression in subcutaneous tumors of mouse and CRC tissues by Immunofluorescence. The stained sections were observed under a fluorescent microscope and photographed. Scale bar = 50 μm. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Inhibition, Over Expression, Expressing, Immunofluorescence, Staining, Microscopy

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: FAO/ACLY signaling promoted Nanog expression via P300/H3K27Ac regulation. Then, Nanog upregulation increased the transcription of P21, P27,COUP-TF1, and hDEC2, and then induced cellular dormancy of serum deprived CRC cells.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A The expression levels of H3K27Ac were analyzed by Western blotting in normal cells, in SF CRC cells and in SF CRC cells treated with ETO. B The interaction of H3K27Ac and Nanog promoter in SF CRC cells was assessed by CHIP-PCR. IgG was used as negative control. Scheme of the different positions of ChIP-PCR primers was also shown (P1: −1844/−823 bp, P2: −1043/−1024 bp, P3: −791/−772 bp, P4: −259/−236 bp). C The expression levels of histone acetyltransferases ( P300, KAT2A, KAT3A ) and histone deacetylases ( HDAC1, HDAC2 ) were examined by PCR in normal HT29, in SF HT29 cells and in SF HT29 cells treated with ETO. D The expression level of P300 was analyzed by Western blotting in normal CRC cells, in serum-deprived CRC cells and in serum-deprived CRC cells treated with ETO. E , F P300 inhibition downregulated the expression of Nanog and H3K27Ac. The serum-deprived HT29 and HCT116 cells were treated with C646 for 24 h. Then, the cells were tested for Nanog expression by PCR (E) and Nanog and H3K27Ac expression by Western blotting (F) . C646: P300 inhibitor. Data are shown as the means ± s.e.m., n = 3; ns: no statistical significance, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A – C The expression of ACLY was analyzed by PCR (A) , Western blotting (B) and Immunofluorescence staining (C) in normal CRC cells, in SF CRC cells and in SF CRC cells treated with ETO. Scale bar = 20 μm. D The expression of ACLY in cytoplasm and nucleus was analyzed by Western blotting. E , F ACLY knockdown decreased the expression of P300 and Nanog. Serum deprived HT29 cells were transfected with ACLY siRNAs or negative control siRNAs. The expression of ACLY , P300 and Nanog was analyzed by PCR. F Western blotting analysis of P300, H3K27Ac and Nanog expression by ACLY inhibition in serum-deprived CRC cells. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: FAO/ACLY signaling promoted Nanog expression via P300/H3K27Ac regulation. Then, Nanog upregulation increased the transcription of P21, P27,COUP-TF1, and hDEC2, and then induced cellular dormancy of serum deprived CRC cells.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing

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: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A Calcein-AM/PI staining of serum-deprived CRC cells. HT29 cells were cultured in serum-free conditions for 12 h (SF12h) or 24 h (SF12h). Cells in normal conditions (NC) were used as controls. Living cells were stained by Calcein-AM (green) while dead cells were visualized by PI (red). The stained cells were observed under a fluorescence microscope and photographed. Scale bar = 50 μm. B Reversible G0-G1 arrest in CRC cells. CRC cells were cultured in normal condition (NC), serum withdrawal for 12h (SF12h) or 24 h (SF24h), Serum replenishment for 12 h (SF + N12h) or 24 h (SF + N24h) after serum withdrawal 24 h, respectively. The cell cycle was analyzed by FCM. C Serum deprivation protected colon cells from chemotherapeutic apoptosis. CRC cells were serum deprived for 24h before treating with 150 μg/ml 5-FU for 18 h. Then, the cells were tested for apoptosis by FCM. D SF stimulation reduced the glucose consumption of CRC cells. HT29 and HCT116 CRC cells were cultured in normal condition or serum-free condition, the culture medium was collected after 24 h. Glucose concentration in the culture medium was measured using Glucose detecting kit and glucose consumption was calculated. E The mRNA expression of genes ( HK2 , PDK1 , PFK1 and PFM ) involved in glucose catabolism in NC and SF HT29 cells was analyzed by PCR. F The protein expression of COUP-TF1, hDEC2, P21 and P27 in NC and SF CRC cells was analyzed by Western blotting. The relative expression was quantified by Image J. G Immunofluorescence staining of COUP-TF1 or hDEC2 and Ki67 from serum-deprived CRC cells or normal cells. Scale bar = 20 μm. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. NC normal condition, SF: serum deprivation for 24 h; FCM Flow cytometry. Data represented at least 3 independent experiments.

Article Snippet: After permeabilizing with 0.1% Triton-X-PBS and blocking in 1% BSA-PBS, the cells were incubated for 1 h with primary antibodies: COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), Nanog (Proteintech #14295-1-AP, CN), or Ki67 (Proteintech #27309-1-AP, CN), washed with PBS for 3 times, incubated with CY3-conjugated anti-rabbit secondary antibody (Servicebio #G1223, CN) for 45 min, and washed with PBS for 3 times. nuclei were marked by DAPI (Servicebio #G1012, CN).

Techniques: Staining, Cell Culture, Fluorescence, Microscopy, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A – C The expression of ACLY was analyzed by PCR (A) , Western blotting (B) and Immunofluorescence staining (C) in normal CRC cells, in SF CRC cells and in SF CRC cells treated with ETO. Scale bar = 20 μm. D The expression of ACLY in cytoplasm and nucleus was analyzed by Western blotting. E , F ACLY knockdown decreased the expression of P300 and Nanog. Serum deprived HT29 cells were transfected with ACLY siRNAs or negative control siRNAs. The expression of ACLY , P300 and Nanog was analyzed by PCR. F Western blotting analysis of P300, H3K27Ac and Nanog expression by ACLY inhibition in serum-deprived CRC cells. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Knockdown, Transfection, Negative Control, Inhibition

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A , B Dormancy markers could be inhibited by ACLY inhibition and reversed by Nanog overexpression in SF HT29 cells. C , D Dormancy markers could be inhibited by P300 inhibition and reversed by Nanog overexpression in SF HT29 cells. E CD31 (green) and Nanog (red) expression in subcutaneous tumors of mouse and CRC tissues by Immunofluorescence. The stained sections were observed under a fluorescent microscope and photographed. Scale bar = 50 μm. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Inhibition, Over Expression, Expressing, Immunofluorescence, Staining, Microscopy

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: FAO/ACLY signaling promoted Nanog expression via P300/H3K27Ac regulation. Then, Nanog upregulation increased the transcription of P21, P27,COUP-TF1, and hDEC2, and then induced cellular dormancy of serum deprived CRC cells.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing

Journal: Cancers

Article Title: NR2F1 Regulates TGF-β1-Mediated Epithelial-Mesenchymal Transition Affecting Platinum Sensitivity and Immune Response in Ovarian Cancer

doi: 10.3390/cancers14194639

Figure Lengend Snippet: Silencing NR2F1 inhibits EMT. ( a ) Western blot analysis of NR2F1, E-cadherin, N-cadherin and vimentin in siNR2F1-transfected A2780 and SKOV-3 cells. The uncropped blots are shown in . ( b – d ) Examination of the invasiveness of siNR2F1-transfected A2780 and SKOV3 cells without or with cisplatin treatment (20 μM) using transwell assay. ( e – h ) Examination of the migration of siNR2F1-transfected A2780 and SKOV3 cells without or with cisplatin treatment (20 μM) using wound-healing assay. CDDP, cisplatin; * p < 0.05; ** p < 0.01.

Article Snippet: Afterwards, membranes were incubated with anti-NR2F1 (Cat NO: 24573-1-AP, Proteintech, Rosemont, IL, USA), TGF-β1 (Cat NO: 21898-1-AP, Proteintech, USA), E-cadherin (Cat NO: 20874-1-AP, Proteintech, USA), N-cadherin (Cat NO: 22018-1-AP, Proteintech, USA), and vimentin (Cat NO: 10366-1-AP, Proteintech, USA) primary antibodies with a dilution of 1:1000 ( v / v ).

Techniques: Western Blot, Transfection, Transwell Assay, Migration, Wound Healing Assay

Journal: Cancers

Article Title: NR2F1 Regulates TGF-β1-Mediated Epithelial-Mesenchymal Transition Affecting Platinum Sensitivity and Immune Response in Ovarian Cancer

doi: 10.3390/cancers14194639

Figure Lengend Snippet: NR2F1 induced epithelial to mesenchymal transition (EMT) via upregulating TGF-β1 expression. ( a , b ) Assessment of TGF-β1 expression in siNR2F1-transfected A2780 and SKOV-3 cells using RT-PCR and western blot. ( c , d ) SiNR2F1-transfected A2780 and SKOV-3 cells were treated with TGF-β1 (5 ng/mL) or without TGF-β1, and protein expression of NR2F1, TGF-β1, E-cadherin, N-cadherin, and vimentin was determined through western blot. The uncropped blots are shown in . ( e – g ) After cisplatin (20 μM) treatment, siNR2F1-transfected A2780 and SKOV-3 cells were treated with TGF-β1 (5 ng/mL) or without TGF-β1, and the invasiveness was examined using transwell assay. ( h – k ) After cisplatin (20 μM) treatment, siNR2F1-transfected A2780 and SKOV-3 cells were treated with TGF-β1 (5 ng/mL) or without TGF-β1, and the migration was examined using wound-healing assay. CDDP, cisplatin; * p < 0.05; ** p < 0.01.

Article Snippet: Afterwards, membranes were incubated with anti-NR2F1 (Cat NO: 24573-1-AP, Proteintech, Rosemont, IL, USA), TGF-β1 (Cat NO: 21898-1-AP, Proteintech, USA), E-cadherin (Cat NO: 20874-1-AP, Proteintech, USA), N-cadherin (Cat NO: 22018-1-AP, Proteintech, USA), and vimentin (Cat NO: 10366-1-AP, Proteintech, USA) primary antibodies with a dilution of 1:1000 ( v / v ).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transwell Assay, Migration, Wound Healing Assay

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A Calcein-AM/PI staining of serum-deprived CRC cells. HT29 cells were cultured in serum-free conditions for 12 h (SF12h) or 24 h (SF12h). Cells in normal conditions (NC) were used as controls. Living cells were stained by Calcein-AM (green) while dead cells were visualized by PI (red). The stained cells were observed under a fluorescence microscope and photographed. Scale bar = 50 μm. B Reversible G0-G1 arrest in CRC cells. CRC cells were cultured in normal condition (NC), serum withdrawal for 12h (SF12h) or 24 h (SF24h), Serum replenishment for 12 h (SF + N12h) or 24 h (SF + N24h) after serum withdrawal 24 h, respectively. The cell cycle was analyzed by FCM. C Serum deprivation protected colon cells from chemotherapeutic apoptosis. CRC cells were serum deprived for 24h before treating with 150 μg/ml 5-FU for 18 h. Then, the cells were tested for apoptosis by FCM. D SF stimulation reduced the glucose consumption of CRC cells. HT29 and HCT116 CRC cells were cultured in normal condition or serum-free condition, the culture medium was collected after 24 h. Glucose concentration in the culture medium was measured using Glucose detecting kit and glucose consumption was calculated. E The mRNA expression of genes ( HK2 , PDK1 , PFK1 and PFM ) involved in glucose catabolism in NC and SF HT29 cells was analyzed by PCR. F The protein expression of COUP-TF1, hDEC2, P21 and P27 in NC and SF CRC cells was analyzed by Western blotting. The relative expression was quantified by Image J. G Immunofluorescence staining of COUP-TF1 or hDEC2 and Ki67 from serum-deprived CRC cells or normal cells. Scale bar = 20 μm. Data are shown as the means ± s.e.m., n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001. NC normal condition, SF: serum deprivation for 24 h; FCM Flow cytometry. Data represented at least 3 independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Staining, Cell Culture, Fluorescence, Microscopy, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A The mRNA expression of Nanog , CD44 and CD133 in NC and SF CRC cells by PCR. B , C Nanog expression in NC and SF CRC cells was tested by Western blotting ( B ) and Immunofluorescence staining ( C ). Scale bar = 20 μm. D Silencing Nanog decreased the cell viability of SF CRC cells. CRC cells were transfected with Nanog siRNAs or negative control siRNAs for 48 h before serum deprivation. Cell viability was detected by CCK8 and marked as SF 0–96 h. E , F Silencing Nanog relieved the G0-G1 arrest (E) and increased the apoptosis rate (F) in SF CRC cells. CRC cells were transfected with Nanog siRNAs or negative control siRNAs for 48 h, and serum deprived for 12 h successively, then collected to analyze in the FCM. G , H Nanog knockdown decreased the expression of COUP-TF1, hDEC2, P21 and P27. Data are shown as the means ± s.e.m., n = 3; ns: no statistical significance, ** P < 0.01, *** P < 0.001. Data represented at least 3 independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Transfection, Negative Control, Knockdown

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A Nanog overexpression increased the cell viability of SF CRC cells. CRC cells were transfected with pCMV3-Nanog plasmids or empty vector for 48 h before serum deprivation. Cell viability was detected by CCK8 and marked as SF 0–96 h. B Nanog overexpression increased the percentage of CRC cells in G0-G1 phase. CRC cells were transfected with pCMV3-Nanog plasmids or empty vector for 48 h before analyzing in FCM. C , D Nanog overexpression increased the expression of COUP-TF1, hDEC2, P21 and P27. CRC cells were transfected with pCMV3-Nanog plasmids for 48 h. The empty vector pCMV3 plasmids were used as controls. The expression of COUP-TF1, hDEC2, P21, and P27 was tested by PCR and Western blotting. The relative expression was quantified by Image J. E SF HT29 cells were collected for ChIP-PCR with anti-Nanog antibody and specific primers for P21 and P27. IgG was used as negative control. Scheme of the different positions of ChIP-PCR primers was also shown (P21-p1: −1366/−1347 bp, P21-p2: −420/−399 bp, P27-p1: −977/ −958 bp, P27-p2: −698/−679 bp). Data are shown as the means ± s.e.m., n = 3; ** P < 0.01, *** P < 0.001. Data represented at least 3 independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: A ETO could block Nanog expression in serum-deprived CRC cells while 2-DG and BPTES could not. The CRC cells were serum-deprived and treated with 100 μM ETO, 10 μM 2-DG or 10 μM BPTES for 24 h. Then, the cells were tested for Nanog expression by PCR. B , C Nanog expression in serum-deprived CRC cells treated with 100 μM ETO was tested by Western blotting ( B ) and Immunofluorescence staining ( C ). The stained cells were observed with a fluorescent microscope and photographed. Scale bar = 20 μm. D ETO treatment decreased the percentage of SF CRC cells in G0-G1 phase. CRC cells were serum deprived and treated with ETO for 24 h before collected to cell cycle analysis. (E) ETO treatment increased the apoptosis rate in SF CRC cells. CRC cells were serum deprived and treated with 100 μM ETO for 24 h, concurrently, and then collected to analyze in the FCM. F ETO could block the expression of COUP-TF1, hDEC2, P21 and P27 in SF CRC cells while 2-DG and BPTES could not. G , H The blockage of dormancy markers by ETO could be reversed by Nanog overexpression. ETO: etomoxir, FAO inhibitor; 2-DG: 2-Deoxy-D-glucose, glycolysis inhibitor; BPTES: glutaminase inhibitor. Data are shown as the means ± s.e.m., n = 3; ns: no statistical significance, * P < 0.05, ** P < 0.01, *** P < 0.001. Data shown represented three independent experiments.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Blocking Assay, Expressing, Western Blot, Immunofluorescence, Staining, Microscopy, Cell Cycle Assay, Over Expression

Journal: Cell Death & Disease

Article Title: Nanog mediated by FAO/ACLY signaling induces cellular dormancy in colorectal cancer cells

doi: 10.1038/s41419-022-04606-1

Figure Lengend Snippet: FAO/ACLY signaling promoted Nanog expression via P300/H3K27Ac regulation. Then, Nanog upregulation increased the transcription of P21, P27,COUP-TF1, and hDEC2, and then induced cellular dormancy of serum deprived CRC cells.

Article Snippet: The primary antibodies used in this study included: GAPDH (Proteintech #10494-1-AP, Wuhan, CN), Nanog (Proteintech #14295-1-AP, CN), ACLY (Proteintech #15421-1-AP, CN), P300 (Cell Signaling Technology #86377, Boston, USA), COUP-TF1 (Proteintech #24573-1-AP, CN), hDEC (Proteintech #212688-1-AP, CN), P21 (Proteintech #10355-1-AP, CN), P27 (Proteintech #25614-1-AP, CN) and H3K27Ac (Cell Signaling Technology #8173, USA).

Techniques: Expressing