Journal: PLOS ONE

Article Title: PHF2 regulates sarcomeric gene transcription in myogenesis

doi: 10.1371/journal.pone.0301690

Figure Lengend Snippet: (A) Phf2 mRNA expression during differentiation in C2C12 myotubes (n = 4 wells per condition in one experiment). (B) Immunofluorescence staining of PHF2 (green) and DAPI (blue) in C2C12 myotubes on days 0 and 7 post differentiation. Scale bar, 100 μm.

Article Snippet: The primary antibodies used for staining were anti-PHF2 antibody (3497S, CST, Boston, MA, USA, 1:100) and anti-Myosin Heavy Chain (MyHC) antibody (MF20 clone, MAB4470, R&D Systems, Minneapolis, MN, USA, 1:100), with incubation overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Staining

Journal: PLOS ONE

Article Title: PHF2 regulates sarcomeric gene transcription in myogenesis

doi: 10.1371/journal.pone.0301690

Figure Lengend Snippet: (A) Overview of the generation of Phf2 KO C2C12 cells. (B) Myoblasts expressing ZsGreen were obtained via cell sorting. Sorted untransfected C2C12 cells (without fluorescence) were used as negative controls. Phf2 KO and mock cells were sorted under the same conditions. Sorted cells are indicated in the orange areas. (C) Protein levels of PHF2 were determined using the Simple Western System.

Article Snippet: The primary antibodies used for staining were anti-PHF2 antibody (3497S, CST, Boston, MA, USA, 1:100) and anti-Myosin Heavy Chain (MyHC) antibody (MF20 clone, MAB4470, R&D Systems, Minneapolis, MN, USA, 1:100), with incubation overnight at 4°C.

Techniques: Expressing, FACS, Fluorescence, Simple Western

Journal: PLOS ONE

Article Title: PHF2 regulates sarcomeric gene transcription in myogenesis

doi: 10.1371/journal.pone.0301690

Figure Lengend Snippet: (A) Phase-contrast microscopy images of mock and Phf2 KO cells 7 d post differentiation. Scale bar, 100 μm. (B) Myotube diameter in mock and Phf2 KO cells 7 d post differentiation (n = 100 cells per group in one experiment). (C) Immunofluorescence staining of Myosin Heavy Chain (MyHC, red) and nuclei (blue). (D) Total MyHC-expressing area (n = 8, cells per group in one experiment). (E) Myotube fusion index based on the ratio of MyHC-nuclei to total nuclei (n = 10, cells per group in one experiment).

Article Snippet: The primary antibodies used for staining were anti-PHF2 antibody (3497S, CST, Boston, MA, USA, 1:100) and anti-Myosin Heavy Chain (MyHC) antibody (MF20 clone, MAB4470, R&D Systems, Minneapolis, MN, USA, 1:100), with incubation overnight at 4°C.

Techniques: Microscopy, Immunofluorescence, Staining, Expressing

Journal: PLOS ONE

Article Title: PHF2 regulates sarcomeric gene transcription in myogenesis

doi: 10.1371/journal.pone.0301690

Figure Lengend Snippet: (A) Principal components analysis with RNA-seq analysis of Phf2 control cells and Phf2 KO cells (n = 3 wells in each group in one experiment). (B) Volcano plot of the RNA-seq data obtained from Phf2 KO cells 2 d post differentiation. (C) Heatmap visualizing the expression profiles based on RNA-seq analysis of Phf2 control and Phf2 KO cells (n = 3 wells per group). (D) KEGG pathway analysis of differentially expressed genes (DEGs) in Phf2 KO C2C12 cells. Numbers in parentheses indicate the number of genes associated with each enriched term. (E) BP DIRECT analysis of DEGs in Phf2 KO C2C12 cells. Numbers in parentheses indicate the number of genes associated with each enriched term.

Article Snippet: The primary antibodies used for staining were anti-PHF2 antibody (3497S, CST, Boston, MA, USA, 1:100) and anti-Myosin Heavy Chain (MyHC) antibody (MF20 clone, MAB4470, R&D Systems, Minneapolis, MN, USA, 1:100), with incubation overnight at 4°C.

Techniques: RNA Sequencing, Control, Expressing

Journal: PLOS ONE

Article Title: PHF2 regulates sarcomeric gene transcription in myogenesis

doi: 10.1371/journal.pone.0301690

Figure Lengend Snippet: (A) mRNA expression of Mef2c , Mybpc2 , Myh7 , Myh2 , Myh1 , Myh4 , Mylk2 , and Tnnt2 in mock and Phf2 KO cells based on qRT-PCR (n = 4 wells per condition in one experiment). (B) CUT&RUN-qPCR analysis of H3K9me2 mark in the promoter region of Mef2c , Mybpc2 , and Myh7 using mock and Phf2 -KO cells (n = 3 wells per condition in one experiment). The amplicon positions from TSS were indicated in parentheses. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with mock C2C12 myotubes.

Article Snippet: The primary antibodies used for staining were anti-PHF2 antibody (3497S, CST, Boston, MA, USA, 1:100) and anti-Myosin Heavy Chain (MyHC) antibody (MF20 clone, MAB4470, R&D Systems, Minneapolis, MN, USA, 1:100), with incubation overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Amplification

Journal: Scientific Reports

Article Title: HIF-1α-mediated augmentation of miRNA-18b-5p facilitates proliferation and metastasis in osteosarcoma through attenuation PHF2

doi: 10.1038/s41598-022-13660-w

Figure Lengend Snippet: Hypoxia-driven HIF-1α is critical for upregulation of miR-18b-5p in OS. ( a ) The luciferase reporter assay accessed luciferase activity of MG-63/143b cells transfected with luciferase reporter plasmid carrying miR-18b promoter sequence or scrambled vector under normoxia and hypoxia. ( b ) RT-qPCR assay showed miR-18b-5p expression in MG-63/143b cells cultured under hypoxia for 0 h, 6 h, 12 h and 24 h. ( c ) (Up) Schematic diagram showed the predicted HRE site on the promoter of miR-18b. (Down) ChIP assay using antibodies against HIF-1α or IgG were performed to validate the binding of HIF-1α on the promoter of miR-18b. ( d ) The luciferase reporter assay accessed luciferase activity of MG-63 and 143b cells transfected with luciferase reporter plasmid carrying WT- miR-18b or MUT- miR-18b, siRNA targeting HIF-1α and negative control under normoxia or hypoxia. ( e ) Western blotting assay accessed protein level of HIF-1α in MG-63/143b cells transfected with siRNA targeting HIF-1α and negative control under normoxia or hypoxia. ( f,g ) RT-qPCR assay showed the expression of miR-18b-5p and PHF2 mRNA in MG-63/143b cells transfected with siRNA targeting HIF-1α and negative control under normoxia or hypoxia.

Article Snippet: Incubated the membrane in 5% non-fat milk by shaking on a shaker for 1 h. Subsequently, the primary antibodies against PHF2 (Abcam, ab124434) and HIF-1α (cell signaling technology, #36169) were incubated with membranes overnight at 4 °C.

Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Sequencing, Quantitative RT-PCR, Expressing, Cell Culture, Binding Assay, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: HIF-1α-mediated augmentation of miRNA-18b-5p facilitates proliferation and metastasis in osteosarcoma through attenuation PHF2

doi: 10.1038/s41598-022-13660-w

Figure Lengend Snippet: Hypoxia induced miR-18b-5p promotes OS via HIF-1α-miR-18b-5p-PHF2 axis. ( a – c ) Pearson correlation analysis showed the correlation between miR-18b-5p and HIF-1α, miR-18b-5p and PHF2, PHF2 and HIF-1α in OS tissues determined by RT-qPCR. ( d ) Schematic diagram showed the molecular mechanism of miR-18b-5p involved in OS.

Article Snippet: Incubated the membrane in 5% non-fat milk by shaking on a shaker for 1 h. Subsequently, the primary antibodies against PHF2 (Abcam, ab124434) and HIF-1α (cell signaling technology, #36169) were incubated with membranes overnight at 4 °C.

Techniques: Quantitative RT-PCR

Journal: Scientific Reports

Article Title: HIF-1α-mediated augmentation of miRNA-18b-5p facilitates proliferation and metastasis in osteosarcoma through attenuation PHF2

doi: 10.1038/s41598-022-13660-w

Figure Lengend Snippet: The sequence of PCR primers.

Article Snippet: Incubated the membrane in 5% non-fat milk by shaking on a shaker for 1 h. Subsequently, the primary antibodies against PHF2 (Abcam, ab124434) and HIF-1α (cell signaling technology, #36169) were incubated with membranes overnight at 4 °C.

Techniques: Sequencing