Journal: Nucleic Acids Research

Article Title: Polyphosphate as a novel regulator of super-enhancer complexes: disruption of phase separation and gene expression

doi: 10.1093/nar/gkag530

Figure Lengend Snippet: Polyphosphate abolishes the nuclear localization and phase separation of YY1. ( A ) Domain graphs of YY1 TAD; IDRs prediction based on ANCHOR2 and IUPred2 algorithms. Scores >0.5 indicate disorder. ( B ) Coomassie-stained NuPAGE analysis of purified MBP-GFP tagged YY1 TAD modified by polyP 700 . ( C ) Western blot analysis after SDS-@PAGE using the YY1 antibody with β-actin as a loading control (n = 3). ( D ) Western blot analysis after SDS-@PAGE of fractions from mCherry or mCherry-PPK1 expressing HeLa cells with antibodies against proteins localized to specific fractions: cytoplasm (Cy) and nuclear (Nu) (n = 3). ( E ) Quantification of changes in cytoplasmic and nuclear protein levels of YY1 relative to β-actin from panel (D). ( F ) Western blot analysis after NuPAGE using the YY1 antibody (n = 3). ( G ) YY1 nuclear speckle formation in HeLa cells were visualized by Confocal microscopy. HeLa cells were co-transfected with GFP-YY1 (first column, green) and either mCherry or mCherry-PPK1 (second column, red); followed by DAPI staining (third column, blue) using ProLong Diamond Antifade Mountant with DAPI (n = 3). ( H ) YY1 droplets formation in HeLa cells were visualized by Confocal microscopy. PolyP was added to the fixed GFP-YY1 expressing (first column, green) HeLa cells, followed by DAPI staining (second column, blue) (n = 3). ( I ) YY1 nuclear speckle formation in HeLa cells was visualized by Confocal microscopy. HeLa cells were co-transfected with either mCherry or mCherry-PPK1 (first column, red) and GFP-PPX (second column, green), followed by DAPI staining (third column, blue) (n = 3). ( J ) Coomassie-stained NuPAGE analysis of polyP 700 modification of purified MBP-mCherry tagged YY1 with the indicated concentration of salt (n = 3). ( K ) phase separation of purified MBP-mCherry tagged YY1 with and without polyP 700 under the indicated concentration of salt. Tubes containing MBP-mCherry tagged YY1 in the buffer containing PEG-8000 (n = 3).

Article Snippet: The YY1 filter plate assay kit was purchased from Signosis (#FA-0006), and assay was conducted as prescribed by Signosis instructions with 15 μM of purified proteins and indicated concentration of polyP 700 or polyP 100 in the reaction mix.

Techniques: Staining, Purification, Modification, Western Blot, SDS Page, Control, Expressing, Confocal Microscopy, Transfection, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Polyphosphate as a novel regulator of super-enhancer complexes: disruption of phase separation and gene expression

doi: 10.1093/nar/gkag530

Figure Lengend Snippet: Polyphosphate inhibits YY1’s recruitment of BRD4-PIR and MED1-PIR and impairs YY1 dimerization and DNA looping function. PolyP 700 affects the droplet formation of BRD4-PIR ( A ), MED1-PIR ( B ) and YY1 (A, B); and the ability of YY1 droplets to incorporate BRD4-PIR (A) or MED-PIR (B) proteins in vitro . MBP-GFP or MBP-mCherry fusion proteins were mixed in buffer containing PEG-8000 and 100 mM NaCl. Indicated fluorescence channels are presented for each mixture (n = 3). ( C ) Model depicting co-immunoprecipitation assay to detect YY1 dimerization with and without the addition of polyP 700 in the resin mixture. Created in BioRender. Jin, J. (2026) https://BioRender.com/r02a397 . ( D ) Western blot analysis after SDS-@PAGE showing the ability of polyP 700 to disrupt co-immunoprecipitation of FLAG-tagged YY1 and HA-tagged YY1 proteins from nuclear lysates prepared from transfected cells using antibodies against FLAG or HA (n = 3). ( E ) Model depicting the in vitro DNA circularization assays used to detect YY1-mediated DNA looping interactions with/without the addition of polyP 700 . Created in BioRender. Jin, J. (2026) https://BioRender.com/o44s370 . ( F ) Results of the in vitro DNA circularization assay visualized by gel electrophoresis showing the ability of polyP 700 to disrupt YY1-mediated DNA loop formation.

Article Snippet: The YY1 filter plate assay kit was purchased from Signosis (#FA-0006), and assay was conducted as prescribed by Signosis instructions with 15 μM of purified proteins and indicated concentration of polyP 700 or polyP 100 in the reaction mix.

Techniques: In Vitro, Fluorescence, Co-Immunoprecipitation Assay, Western Blot, SDS Page, Immunoprecipitation, Transfection, Nucleic Acid Electrophoresis

Journal: Nucleic Acids Research

Article Title: Polyphosphate as a novel regulator of super-enhancer complexes: disruption of phase separation and gene expression

doi: 10.1093/nar/gkag530

Figure Lengend Snippet: Polyphosphate inhibits YY1’s binding to DNA and affects gene regulation. ( A ) EMSA results using 4% native gel showing the binding of MBP-mCherry tagged YY1 to IR700Dye-labled YY1 binding motif after polyP 700 treatment. ( B ) Schematic of the filter plate method used for assessing YY1 binding to its biotinylated DNA target. Created in BioRender. Jin, J. (2026) https://BioRender.com/s56k283 . Luminescent quantification of purified MBP-mCherry tagged YY1 binding to target DNA via the filter plate assay after treatment of 5 mM polyP 700 ( C ) or the indicated concentration of polyP 700 ( D ). Unpaired t test; ns, P > .05, ∗∗ P ≤ .01, ∗∗∗∗ P ≤ .0001, error bars ± standard deviation (n = 3). ( E ) EMSA results using 4% native gel showing the binding of MBP-mCherry tagged YY1 to IR700Dye-labled human telomere G4 structure after treatment of polyP 700 . ( F ) Fluorescence anisotropy for measuring the binding of MBP-YY1 protein with IR700Dye-labled human telomere G4 structures after polyP 700 treatment. Unpaired t test; ns, P > .05, ∗∗ P ≤ .01, ∗∗∗∗ P ≤ .0001, error bars ± standard deviation (n = 3). Quantification results of mRNA expression levels of MYC ( G ), PDHB ( H ), and MANF ( I ) genes in HeLa cells transfected with plasmids expressing either mCherry or mCherry-PPK1. Unpaired t- test; ns, P > .05, ∗∗ P ≤ .01, ∗∗∗∗ P ≤ .0001, error bars ± standard deviation.

Article Snippet: The YY1 filter plate assay kit was purchased from Signosis (#FA-0006), and assay was conducted as prescribed by Signosis instructions with 15 μM of purified proteins and indicated concentration of polyP 700 or polyP 100 in the reaction mix.

Techniques: Binding Assay, Purification, Concentration Assay, Standard Deviation, Fluorescence, Expressing, Transfection

Journal: Nucleic Acids Research

Article Title: Polyphosphate as a novel regulator of super-enhancer complexes: disruption of phase separation and gene expression

doi: 10.1093/nar/gkag530

Figure Lengend Snippet: Medium chain polyphosphate affects the functions of YY1, BRD4 and MED1. Coomassie-stained NuPAGE analysis showing polyP 100 modification on MBP-mCherry tagged YY1 ( A ), MBP-GFP tagged BRD4 PIR ( B ), and MED1 PIR ( C ). ( D ) Western blot analysis after SDS-@PAGE showing the ability of polyP 100 to impair co-immunoprecipitation of FLAG-tagged YY1 and HA-tagged YY1 proteins from nuclear lysates prepared from transfected cells using antibodies against FLAG or HA (n = 3). ( E ) EMSA results using 4% native gel showing the binding of MBP-mCherry tagged YY1 to IR700Dye-labled YY1 binding motif after polyP 100 treatment. Luminescent quantification of purified MBP-mCherry tagged YY1 binding to target DNA via the filter plate treated with 5 mM polyP 100 ( F ) or the indicated concentration of polyP 100 ( G ). Unpaired t test; ns, P > .05, ∗∗∗∗ P ≤ .0001, error bars ± standard deviation (n = 3). ( H ) EMSA results using 4% native gel showing the binding of MBP-mCherry tagged YY1 to IR700Dye-labled human telomere G4 structure with polyP 100 treatment. ( I ) Fluorescence anisotropy for measuring the binding of MBP-YY1 protein with IR700Dye-labled human telomere G4 structures after treatment with polyP 100 . Unpaired t -test; ns, P > .05, ∗∗ P ≤ .01, error bars ± standard deviation (n = 3).

Article Snippet: The YY1 filter plate assay kit was purchased from Signosis (#FA-0006), and assay was conducted as prescribed by Signosis instructions with 15 μM of purified proteins and indicated concentration of polyP 700 or polyP 100 in the reaction mix.

Techniques: Staining, Modification, Western Blot, SDS Page, Immunoprecipitation, Transfection, Binding Assay, Purification, Concentration Assay, Standard Deviation, Fluorescence

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: YY1 positively regulated the expression of ANXA3 at the transcriptional level. (A) The binding ability of YY1 to the ANXA3 promoter in AC16 cells was detected by ChIP assay, n = 6. (B) The ability of YY1 to active the ANXA3 promoter in HEK293 cells was detected by Dual-luciferase reporter assay, n = 6. (C-D) Relative YY1 and ANXA3 mRNA expression levels in AC16 cells analyzed by RT-qPCR, n = 4. (E-H) Relative YY1 and ANXA3 protein expression levels in AC16 cells analyzed by Western blot, n = 5. The data were presented as the mean ± SD. ∗∗P < 0.01 versus the IgG group or the OE-NC group; ∗∗∗P < 0.001 versus the si-NC group (YY1) or the OE-NC group (YY1); # P < 0.05 versus the OE-NC group (ANXA3); ## P < 0.01 versus the OE-NC group (ANXA3) or the si-NC group (ANXA3).

Article Snippet: After restoring the frozen sections to room temperature, blocked them with 5%bovine serum albumin (w/v)for 30 min, then incubated them with anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:200 [v/v]), anti-YY1 antibody (Proteintech, 22156-1-AP; 1:200 [v/v]), anti-LAMP1 antibody (Proteintech, 21997-1-AP; 1:200 [v/v]) and anti-3-NT antibody (Millipore, Massachusetts, 06284; 1:400 [v/v]) for 16 h respectively.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: T2DM attenuated the function of YY1 by promoting nitrosative stress. (A-B) Relative YY1 protein expression levels in the cardiac tissue of mice analyzed by Western blot, n = 4. (C-D) Relative YY1 protein expression levels in AC16 cells analyzed by Western blot, n = 6. (E-F) Representative images of IF and the statistical chart were used to observe the nuclear translocation situation of YY1 in the cardiac tissue of mice. Bars: 25 μm, n = 4. (G-H) Relative YY1 protein expression levels in the nucleus of AC16 cells analyzed by Western blot, n = 4-5. (I) Representative images of IF were used to observe the expression level of 3-NT in the cardiac tissue of mice. Bars: 25 μm. (J-K) Relative YY1 protein expression levels in the nucleus of AC16 cells analyzed by Western blot, n = 3. The data were presented as the mean ± SD. ∗ P < 0.05 versus the Vehicle group; ∗∗∗ P < 0.001 versus the db/m group; # P < 0.05 versus the HGPA group; ### P < 0.01 versus the HGPA group.

Article Snippet: After restoring the frozen sections to room temperature, blocked them with 5%bovine serum albumin (w/v)for 30 min, then incubated them with anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:200 [v/v]), anti-YY1 antibody (Proteintech, 22156-1-AP; 1:200 [v/v]), anti-LAMP1 antibody (Proteintech, 21997-1-AP; 1:200 [v/v]) and anti-3-NT antibody (Millipore, Massachusetts, 06284; 1:400 [v/v]) for 16 h respectively.

Techniques: Expressing, Western Blot, Translocation Assay

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: Tyr185 site of YY1 nitrated was involved in inhibiting the transcription of ANXA3. (A-B) Relative nitration levels of YY1 in HEK293 cells analyzed by IP, n = 7. (C-D) Relative YY1 expression levels in the nucleus of AC16 cells analyzed by Western blot, n = 5. (E-F) Relative YY1 and ANXA3 mRNA expression levels in AC16 cells analyzed by RT-qPCR, n = 6. The data were presented as the mean ± SD. ∗P < 0.05 versus the WT group; ∗∗P < 0.01 versus the WT group; ∗∗∗P < 0.001 versus the WT group; ## P < 0.01 versus the WT + HGPA group.

Article Snippet: After restoring the frozen sections to room temperature, blocked them with 5%bovine serum albumin (w/v)for 30 min, then incubated them with anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:200 [v/v]), anti-YY1 antibody (Proteintech, 22156-1-AP; 1:200 [v/v]), anti-LAMP1 antibody (Proteintech, 21997-1-AP; 1:200 [v/v]) and anti-3-NT antibody (Millipore, Massachusetts, 06284; 1:400 [v/v]) for 16 h respectively.

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