Journal: Molecules and Cells

Article Title: The Histone Demethylase PHF2 Promotes Fat Cell Differentiation as an Epigenetic Activator of Both C/EBPα and C/EBPδ

doi: 10.14348/molcells.2014.0180

Figure Lengend Snippet: PHF2 knock-down reduced the adipogenesis capacity of 3T3-L1 pre-adipocytes. (A) Oil Red-O staining of stable 3T3-L1 cell lines expressing control or PHF2 shRNA. Cell extracts were analyzed with Western blotting using anti-PHF2 and tubulin antibodies (left). PHF2 mRNA and protein levels of indicated stable cell lines were measured before adipogenesis and plotted (middle panels). Oil Red-O dye was extracted with isopropanol and the optical density at 520 nm (O. D 520 ) was measured (left). Bars represents the means ± SDs of three independent experiments, and “*” denotes P < 0.01 versus the control shRNA (sh-con) group. (B) Number of hits for genes involved in cellular pathways showing reduced expression after adipogenesis in the PHF2 knock-down cell line. Genes repressed by more than 0.5-fold compared to the control cell line and had a p-value of less than 0.05 were selected. A total of 4069 genes were analyzed using the DAVID ( http://david.abcc.ncifcrf.gov/ ) database.

Article Snippet: Antibodies against PPARγ, C/EBPα, C/EBPβ, C/EBPδ, FABP4, HA and beta-tubulin were purchased from SantaCruz Biotech. (USA), and antibody against PHF2 and H3K9-Me2 were from Cell Signaling (USA).

Techniques: Staining, Expressing, shRNA, Western Blot, Stable Transfection

Journal: Molecules and Cells

Article Title: The Histone Demethylase PHF2 Promotes Fat Cell Differentiation as an Epigenetic Activator of Both C/EBPα and C/EBPδ

doi: 10.14348/molcells.2014.0180

Figure Lengend Snippet: PHF2 knock-down reduced mRNA and protein levels of adipogenesis marker genes. (A) 3T3-L1 stable cells were differentiated with adipogenesis-inducing media and then lysed at the indicated times. Relative mRNA levels of the indicated genes were determined by qRT-PCR. Bars represents the means ± SDs of three independent experiments, and “*” denotes p < 0.01 versus the control shRNA (sh-con) group. (B) Control and PHF2 shRNA-expressing stable 3T3-L1 cell lines were treated with adipogenic rea-gent for the indicated number of days (left) or hours (right), and cell lysates were analyzed with Western blotting with the indicated antibodies.

Article Snippet: Antibodies against PPARγ, C/EBPα, C/EBPβ, C/EBPδ, FABP4, HA and beta-tubulin were purchased from SantaCruz Biotech. (USA), and antibody against PHF2 and H3K9-Me2 were from Cell Signaling (USA).

Techniques: Marker, Quantitative RT-PCR, shRNA, Expressing, Western Blot

Journal: Molecules and Cells

Article Title: The Histone Demethylase PHF2 Promotes Fat Cell Differentiation as an Epigenetic Activator of Both C/EBPα and C/EBPδ

doi: 10.14348/molcells.2014.0180

Figure Lengend Snippet: PHF2 physically interacts with C/EBPα and C/EBPδ (A) Flag-SBPPHF2 and HA-tagged C/EBPβ, C/EBPδ, PPARγ and non-tagged C/EBPα were ectopically expressed in HEK293T cells, and cell lysates were incubated with streptavidin beads. The bound proteins were eluted with biotin. Input and streptavidin-bound proteins were detected by western blotting with the indicated antibodies. (B) F/S-tagged PHF2 fragments (1–4) were co-expressed with C/EBPα or HA-C/EBPδ plasmid in HEK293T cells. After F/S-PHF2 fragments were precipitated using streptavidin beads, co-precipitated proteins were detected by Western blotting using the indicated antibodies. (C) C/EBPα and C/EBPδ were co-expressed with wild-type PHF2 or the PHF2-H249A mutant. After HAC/EBPδ was precipitated using an HA antibody, co-precipitated proteins were detected by western blotting using the indicated antibodies.

Article Snippet: Antibodies against PPARγ, C/EBPα, C/EBPβ, C/EBPδ, FABP4, HA and beta-tubulin were purchased from SantaCruz Biotech. (USA), and antibody against PHF2 and H3K9-Me2 were from Cell Signaling (USA).

Techniques: Incubation, Western Blot, Plasmid Preparation, Mutagenesis

Journal: Molecules and Cells

Article Title: The Histone Demethylase PHF2 Promotes Fat Cell Differentiation as an Epigenetic Activator of Both C/EBPα and C/EBPδ

doi: 10.14348/molcells.2014.0180

Figure Lengend Snippet: PHF2 was recruited to PPARG and FABP4 promoters with C/EBPα and C/EBPδ. 3T3-L1 cells expressing control shRNA or PHF2 shRNA were stimulated with an adipogenesis cocktail for the indicated times 24 th , and chromatin immunoprecipitation was performed with anti-PHF2, anti-C/EBPα anti-C/EBPδ or histone H3K9-Me2 antibodies. The resulting immuno-precipitated DNA was analyzed by qPCR using primers for (A) the PPARG promoter sequence, (B) the CEBPA promoter sequence, or (C) the FABP4 promoter sequence. The positions of primers relative to the transcriptional start site are indicated. 3T3L1 pre-adipocytes transfected with (D) C/EBPδ or (E) C/EBPα siRNAs (sequences listed in ) were induced with adipogenesis cocktail for the indicated times. Immunoprecipitated DNAs were PCR-amplified with primers targeting the (D) PPARG promoter or (E) FABP4 promoter CEBP response element. Bars represents the means ± SDs of three independent experiments, and “*” denotes p < 0.01 versus the control group. (F) Schematic illustrating the role of PHF2 in C/EBPδ and C/EBPα transcriptional activation.

Article Snippet: Antibodies against PPARγ, C/EBPα, C/EBPβ, C/EBPδ, FABP4, HA and beta-tubulin were purchased from SantaCruz Biotech. (USA), and antibody against PHF2 and H3K9-Me2 were from Cell Signaling (USA).

Techniques: Expressing, shRNA, Chromatin Immunoprecipitation, Sequencing, Transfection, Immunoprecipitation, Amplification, Activation Assay

Journal: Journal of Pathology and Translational Medicine

Article Title: Implication of PHF2 Expression in Clear Cell Renal Cell Carcinoma

doi: 10.4132/jptm.2017.03.16

Figure Lengend Snippet: Plant homeodomain finger 2 (PHF2) expression in non-neoplastic renal tissue. PHF2 is expressed in the nucleus and cytoplasm of the proximal tubular epithelium and in the nucleus of the distal tubular epithelium and podocytes in the glomerulus. (A) Cortex. (B) Medulla.

Article Snippet: Polyclonal rabbit anti-PHF2 antibody (Novus Biologicals, Littleton, CO, USA) was diluted 1:200.

Techniques: Expressing

Journal: Journal of Pathology and Translational Medicine

Article Title: Implication of PHF2 Expression in Clear Cell Renal Cell Carcinoma

doi: 10.4132/jptm.2017.03.16

Figure Lengend Snippet: Relationship between PHF2 expression and clinicopathological characteristics

Article Snippet: Polyclonal rabbit anti-PHF2 antibody (Novus Biologicals, Littleton, CO, USA) was diluted 1:200.

Techniques: Expressing, Biomarker Discovery

Journal: Journal of Pathology and Translational Medicine

Article Title: Implication of PHF2 Expression in Clear Cell Renal Cell Carcinoma

doi: 10.4132/jptm.2017.03.16

Figure Lengend Snippet: Kaplan-Meier curves. Cancer-specific and progression-free survival according to plant homeodomain finger 2 (PHF2) expression.

Article Snippet: Polyclonal rabbit anti-PHF2 antibody (Novus Biologicals, Littleton, CO, USA) was diluted 1:200.

Techniques: Expressing

Journal: Journal of Pathology and Translational Medicine

Article Title: Implication of PHF2 Expression in Clear Cell Renal Cell Carcinoma

doi: 10.4132/jptm.2017.03.16

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of cancer-specific and progression-free survival

Article Snippet: Polyclonal rabbit anti-PHF2 antibody (Novus Biologicals, Littleton, CO, USA) was diluted 1:200.

Techniques: Biomarker Discovery, Expressing

Journal: bioRxiv

Article Title: The AMPKα2/PHF2 axis is critical for turning over lipid droplets during muscle stem cell fate

doi: 10.1101/2025.01.18.630727

Figure Lengend Snippet: CSA distribution of muscle fibers on cryosections of regenerated TA muscles at 11dpi (A) and 28dpi (B). (C) Representative images of immunostainings. (D) Number of myofibers per mm2 at 28dpi. (E) Quantification of the number of PAX7 pos Ki67 neg cells per mm 2 at 28 dpi. (F) Quantification of the number of MYOG pos cells per mm 2 on CTRL SC and PHF2 SCiKO TA cryosections at 11dpi. (G) Percentage of nuclei per myotube, (H) Percentage of PAX7 pos MYOG neg (yellow arrowhead) and (I) PAX7 neg MYOG pos cells (purple arrowhead) in cultures after 48h in LSM. (J) Representative images of immunostainings. (K) Experimental setup. Fusion index (L) and percentage of nuclei per myotube (M) in cultures after 48h in LSM. (N) Experimental setup. Percentage of nuclei per myotube (O) and of PAX7 pos cells (P) in PHF2 SCiKO cells transfected with either MOCK, PHF2 wt or PHF2 H249A . Scale bars, 50 μm. n = 3-6 mice/genotype. n = 3-8 primary cultures/genotype. Values are mean or percentage mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t -test [D-E-F-H-I-L panels], Sidak’s test after two-way ANOVA [A-B panels], Tukey’s test after one way [G-M-P panels] or two-way ANOVA [O panel]).

Article Snippet: They were transferred in a wet chamber for 1h30 at RT in 1% BSA,50 mM Tris-HCl, pH 8.2 for 1h30 at RT, labelled with gold conjugated Secondary antibody (Goat anti Rabbit 10nm for Myog and Goat anti Rabbit 5nm for Phf2) (Aurion).

Techniques: Muscles, Transfection

Journal: bioRxiv

Article Title: The AMPKα2/PHF2 axis is critical for turning over lipid droplets during muscle stem cell fate

doi: 10.1101/2025.01.18.630727

Figure Lengend Snippet: (A-B) Percentage of MYOG pos BODIPY pos cells transfected with either MOCK, PHF2 wt or PHF2 S655E and PHF2 S655A . Representative images. (C) Percentage of MYOG pos and (D) percentage of MYOG pos BODIPY pos cells after 48h in LSM. (E) Representative images. (F) Percentage of MYOG pos BODIPY pos cells transfected with either MOCK, PHF2 S655E or PHF2 S655A . Scale bars, 5 μm. n = 3 primary cultures/genotype. Values are mean or percentage mean ± SEM. ***P < 0.001 (Student t -test [C-D panels] and Tukey’s test after one way-ANOVA [B-F panels]).

Article Snippet: They were transferred in a wet chamber for 1h30 at RT in 1% BSA,50 mM Tris-HCl, pH 8.2 for 1h30 at RT, labelled with gold conjugated Secondary antibody (Goat anti Rabbit 10nm for Myog and Goat anti Rabbit 5nm for Phf2) (Aurion).

Techniques: Transfection

Journal: bioRxiv

Article Title: The AMPKα2/PHF2 axis is critical for turning over lipid droplets during muscle stem cell fate

doi: 10.1101/2025.01.18.630727

Figure Lengend Snippet: (A) Clustering of EYFP pos -FACS-isolated cells from CTRL SC and PHF2 SCiKO at 11dpi (n=3 mice pooled in 1 sample/genotype). (B) Myog pos myocytes cluster. Plin2, Plin3 (C) and Gdi2 (D) mRNA expression levels. (E) GDI2 protein level in CTRL SC and PHF2 SCiKO mononucleated cells 24h post LSM. (F) Integrative Genomics Viewer (IGV) snapshot depicting PHF2 enrichment (MACS peak call in dark blue) on the Gdi2 promoter region (GSM3462720). PLA analysis (G), electron microscopy analysis of LD-mitochondria contacts (H) and confocal analysis of LD-RAB8a-mitochondria contacts (I) in CTRL SC and PHF2 SCiKO mononucleated cells 24h post LSM. Scale bars, 1, 2 and 5 μm. n = 3 primary cultures/genotype. For (H panel), n=20 cells/genotype/conditions. Values are mean or percentage mean ± SEM. **P < 0.01, ****P < 0.0001 (Student t -test [E panel] and Tukey’s multiple comparison test after one way-ANOVA [G-H-I panels]).

Article Snippet: They were transferred in a wet chamber for 1h30 at RT in 1% BSA,50 mM Tris-HCl, pH 8.2 for 1h30 at RT, labelled with gold conjugated Secondary antibody (Goat anti Rabbit 10nm for Myog and Goat anti Rabbit 5nm for Phf2) (Aurion).

Techniques: Isolation, Expressing, Electron Microscopy, Comparison

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: PHF2 expression and clinicopathologic features of breast cancer a

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: Expressing

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: Down-expression of PHF2 in breast cancer cells and tissues. (A) The expression profile of PHF2mRN from the online database. (1) Breast ( n = 144), (2) ductal breast cancer in situ ( n = 10), (3) tubular breast cancer ( n = 67), (4) medullary breast cancer ( n = 32), (5) mucinous breast cancer ( n = 46), and (6) invasive ductal and lobular breast cancer ( n = 90). (B) The expression of PHF2 mRNA in breast cancer cell lines ( n = 5) was detected by RT-qPCR. (C) The expression of PHF2 mRNA in paired breast cancer tissues ( n = 28) was detected by RT-qPCR. (D) The expression of PHF2 protein in breast cancer tissues ( n = 80) and normal breast tissues ( n = 40): (7) low expression in breast cancer tissues (original magnification, ×400). (8) High expression in normal breast tissues (original magnification, ×400). * P < 0.05 and *** P < 0.001, vs. vector.

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: Expressing, In Situ, Quantitative RT-PCR, Plasmid Preparation

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: The expression of PHF2 between breast cancer and normal breast tissues

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: Expressing

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: A low expression of PHF2 indicates a poor prognosis of breast cancer patients. Data taken from the Kmplot online database http://kmplot.com/analysis/ . PHF2 expression and the over survival and the recurrence-free survival (RFS) in patients with different types of breast cancer.

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: Expressing

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: PHF2 represses the proliferation of breast cancer cells. (A) PHF2 expression levels in MDA-MB-231 and MCF-7 after infection by Ad-RFP (vector) and Ad-PHF2 (PHF2) detected by RT-qPCR. (B) Over-expression of PHF2 markedly diminished the ability of colony formation in MDA-MB-231 and MCF-7. (C) The statistics of the colonies; the number of colonies in the vector group was set to 100%. (D and E) Ectopic expression of PHF2 obviously inhibited the proliferation of MDA-MB-231 and MCF-7 by CCK-8, respectively. (F) PHF2 arrested the cell cycle of MDA-MB-231 mainly in the G1, and G2/M phases. (G) The statistics of the cell cycles. * P < 0.05,** P < 0.01 and *** P < 0.001, vs. vector.

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: Expressing, Infection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, CCK-8 Assay

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: PHF2 inhibits the tumor growth in vivo . (A) Xenograft tumor size in both groups ( n = 4 per group). (B) Growth curve of the tumors. The growth was significantly inhibited after 2 weeks of the injected Ad-PHF2 in the tumor. (C) Final weight of the xenograft tumors in both groups. * P < 0.05, *** P < 0.001, vs. vector.

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: In Vivo, Injection, Plasmid Preparation

Journal: RSC Advances

Article Title: The expression and biological function of the PHF2 gene in breast cancer

doi: 10.1039/c8ra06017g

Figure Lengend Snippet: Molecular functional pathways and process of PHF2.

Article Snippet: Next, the sections were incubated with the primary antibody PHF2 (HPA010831, 1 : 250), which was produced in rabbits and was purchased from Sigma (USA), overnight at 4 °C.

Techniques: Functional Assay

Journal: Oncotarget

Article Title: The prognostic significance of nuclear expression of PHF2 and C/EBPα in clear cell renal cell carcinoma with consideration of adipogenic metabolic evolution

doi: 10.18632/oncotarget.19949

Figure Lengend Snippet: Clinicopathologic features of patients with ccRCC and correlation between nuclear PHF2 and C/EBPα expression and clinicopathologic parameters.

Article Snippet: Each polyclonal rabbit anti-PHF2 antibody (Novus Biologicals, Littleton, CO) and polyclonal rabbit anti-C/EBPα antibody (Santa Cruz Biotechnology, Dallas, TX) was diluted 1:200.

Techniques: Expressing

Journal: Oncotarget

Article Title: The prognostic significance of nuclear expression of PHF2 and C/EBPα in clear cell renal cell carcinoma with consideration of adipogenic metabolic evolution

doi: 10.18632/oncotarget.19949

Figure Lengend Snippet: Multivariate analysis of cancer-specific and progression-free survival with PHF2 and C/EBPα nuclear expression in 344 patients with ccRCC (Cox proportional hazard model).

Article Snippet: Each polyclonal rabbit anti-PHF2 antibody (Novus Biologicals, Littleton, CO) and polyclonal rabbit anti-C/EBPα antibody (Santa Cruz Biotechnology, Dallas, TX) was diluted 1:200.

Techniques: Expressing

Journal: Nature Communications

Article Title: The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity

doi: 10.1038/s41467-018-04361-y

Figure Lengend Snippet: Liver-specific Phf2 overexpression causes quick-onset hepatosteatosis. a – h Mice, injected with either GFP or Phf2 overexpressing adenovirus, were studied 3 weeks later in the fed state. a Phf2 mice develop hepatic steatosis as shown by increased liver size and by oil red O staining of liver sections. Scale bars = 100 μm ( n = 10 per group). b Measurement of PKCε activity ( n = 6 per group). c Liver DAG content in the cytosol and at the plasma membrane ( n = 8 per group). d Relative expression of liver pro-inflammatory genes ( n = 10 per group). e Insulin tolerance test ( n = 10 per group). f Oral glucose tolerance test and insulin levels during the OGTT test ( n = 10 per group). g Western blot analysis of the PI3K/Akt signaling pathway in liver ( n = 20 per group). h Heatmap visualization of relative SFA, MUFA, and PUFA content in liver DAG, TG, and cholesterol ester species ( n = 10 per group). i – k Isolated primary hepatocytes overexpressing Phf2 and in which SCD1 expression was inhibited were incubated in the presence of palmitate (480 μM) for 24 h. i Representative western blots of the pro-inflammatory signaling pathway ( n = 4). j Percentage of hepatocyte SFA, MUFA and PUFA content in indicated culture conditions ( n = 3). k Representative western blot analysis of the PI3K/Akt signaling ( n = 4). All error bars represent mean ± SEM. Statistical analyses were made using unpaired t -test. * P < 0.01 GFP compared to Phf2, ** P < 0.05 GFP compared to Phf2. # P < 0.01 NCD compared to HFHS

Article Snippet: PKCε was immunoprecipitated from liver samples of GFP or Phf2 overexpressing mice using 0.5 μg of polyclonal rabbit anti PKCε antibody (Santa Cruz, CA).

Techniques: Over Expression, Injection, Staining, Activity Assay, Clinical Proteomics, Membrane, Expressing, Western Blot, Isolation, Incubation