Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 (panels a and b), and then were induced to differentiate in MDM for 4 (panels c,d,i,j) or 6 days (panels e and f and panels g and h). At the different time points cells were fixed and incubated with a monoclonal antibody against Troponin-T (panels a to f) or stained with Oil-red-O (panels g and h). At day 4, cells were also analyzed with a monoclonal antibody against Fabp4/aP2 (panels i and j). Cells were observed at a magnification of 20x, ( __ ): 60 µm. B: At day 1, C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h and then were induced to differentiate in MDM. Cells were then collected at the indicated time points and expression of specific mRNAs was analyzed by RT-PCR amplification. PCR products were analyzed on 1.2% agarose gels. Photographs of the gels are shown. RNase L protein expression was analyzed as in . Autoradiographs of these gels are shown (RNase L-P). C: Quantification of lipids accumulated in C2-RNase L cells treated ( ) or not ( ) with 5 mM IPTG for 6 h and then induced to differentiate in MDM for 6 days. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in untreated C2-RNase L cells was set to 1. Data were expressed as mean±SD of three different experimental points. (*) P<0.01 compared to non-induced cells.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Incubation, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 and then induced to differentiate in ADM. Cells were fixed and stained with Oil-red-O to reveal lipids accumulation at day 0, 3 and 6. Cells were observed at a magnification of 20x, ( __ ): 60 µm. B: Quantification of lipids in control ( ) or IPTG-treated C2-RNase L cells ( ) at day 0, 3 and 6 after induction of differentiation in ADM. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in C2-RNase L cells at T = 0, before addition of IPTG, was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.01 compared to untreated cells at T = 3. C: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 and then induced to differentiate in ADM. Cells were fixed at day 0, 3 and 6 and then incubated with an antibody against Fabp4/aP2 (Red). DNA was stained with Dapi (Blue). Cells were observed at 20x, ( __ ): 50 µm. D: Analysis of mRNA expression by RT-PCR amplification at different time points during adipocyte differentiation. RNase L was induced (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 2 and then cells were switched to ADM to induce adipocyte differentiation. PCR products were analyzed by gel electrophoresis. Photographs of the gels are shown.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Staining, Standard Deviation, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated, or not (0), with 5 mM IPTG for (2) or (6)h. Cells were then harvested and analyzed for RNase L 2-5A binding activity with the 2-5A radio-covalent binding assay. Proteins were separated on 10% polyacrylamide gels. A representative autoradiography of the gel and a densitometry of the gel are shown. The amount of 2-5A binding activity in C2-RNase L cells in the absence of IPTG was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.0 compared to non- induced cells (**) P<0.01 compared to control, untreated cells. B: RNase L was induced by 5 mM IPTG for 0, 2 or 6 hours, and then mRNA expression was analyzed by RT-PCR amplification and agarose gel electrophoresis. Photographs and densitometric analysis of the gels are shown. The level of the different mRNAs in untreated cells was set at 1. Quantification of EEF1α mRNA was used as a control of semi-quantitative RT-PCR experiment. Error bars refer to the standard deviation obtained in three independent experiments. C: C2-RNase L cells were treated, or not, with 5 mM IPTG for 2 or 6 h and then induced to differentiate in ADM. At T = 0, T = 2, T = 4 and T = 6 days after induction of differentiation, cells were fixed and stained with Oil-red-O to reveal lipid accumulation (panel on the right) or incubated with an antibody against Fabp4/aP2 (Red) (panel on the left); DNA was stained with Dapi (Blue). Cells were observed at 20x, ( __ ): 50 µm. D: Quantification of lipids in control or IPTG-treated C2-RNase L cells (for 2 or 6 h) at T = 0, T = 2, T = 4 and T = 6 days after induction of differentiation with ADM. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in C2-RNase L cells at T = 0, before IPTG treatment, was set to 1. Error bars refer to the standard deviation obtained in three independent experimental points. (*) P<0.01 compared to untreated cells at the same time during adipocyte differentiation.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Binding Assay, Activity Assay, Autoradiography, Standard Deviation, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Quantitative RT-PCR, Staining, Incubation

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: RNase L activity in C2-RNase L cells, C2-RNase L cells treated with IPTG and C2-RLI cells. C2-RLI cells (1, ), C2-RNase L cells (2, ) and C2-RNase L treated with 5 mM IPTG (3, ) for 6 h were analyzed for RNase L 2-5A binding activity with the 2-5A radiocovalent binding assay. Proteins were separated in 10% polyacrylamide gels. A representative autoradiography and a densitometry of the gel are shown. The amount of 2-5A binding activity in C2-RLI cells was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. B: C2-RLI differentiation. Cells were fixed and incubated with Oil-red-O to reveal lipid accumulation (i = day 0, j = day 6 after induction of differentiation with ADM) and expression of Troponin T and Fabp4/aP2 was analyzed using monoclonal antibodies against Troponin T (Green, c,d) and Fabp4/aP2 (Red e,f) at day 0 (a,c,e,g,i) and day 6 after induction of differentiation with ADM (b,d,f,h,j). DNA was stained with Dapi (Blue a,b,g,h). A merge of Dapi, Troponin T and Fabp4/aP2 labeling is shown (g,h). Cells were observed at 20x, ( __ ): 20 µm. C: Quantification of lipids in C2-RLI cells at day 0 ( ) and day 6 after induction of differentiation with ADM ( ). A value of 1 corresponds to the amount of lipids in C2-RLI cells at T = 0, before differentiation. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.01 compared to untreated C2-RNase L cells and (**) P<0.01 compared to IPTG-treated C2-RNase L cells at the same time point during adipocyte differentiation. D: Analysis of mRNA expression by RT-PCR amplification at different time points during adipocyte differentiation. C2-RLI cells were switched to ADM to induce adipocyte differentiation at day 1. PCR products were analyzed by gel electrophoresis. Photographs of the gels are shown.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Activity Assay, Binding Assay, Autoradiography, Standard Deviation, Incubation, Expressing, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: Notch signaling is a critical initiator of roof plate formation as revealed by the use of RNA profiling of the dorsal neural tube

doi: 10.1101/2020.12.09.417279

Figure Lengend Snippet: (A-L) In situ hybridization of transverse sections through the neural tube at the level of the forelimb in wildtype (WT) and Wnt1-Cre; Mib1 fl/fl mouse embryos. Math1 expression at E10.5 (A, B) and E11.5 (C, D) showing loss of dI1 population in mutants; Ngn1 expression at E10.5 (E, F) and E11.5 (G, H) and Ngn2 expression at E10.5 (I, J) and E11.5 (K, L) showing dorsal-ward expansion of the dI2 population. (M-X) Immunostaining of transverse sections through the dorsal neural tube at the level of the forelimb in wildtype (WT) and Wnt1-Cre; Mib1 fl/fl mouse embryos. Isl1 expression at E10.5 (M, N) and E11.5 (O, P) showing a dorsal-ward expansion of dI3 interneurons at E11.5; AP2 α expression at E10.5 (Q, R) and E11.5 (S, T) showing a dorsal-ward expansion in mutants at both stages (positive staining within the neural tube lumen in T is due to erythrocyte autofluorescence); Tuj1 expression at E10.5 (U, V) and E11.5 (W, X) showing ectopic expression of the axonal marker Tuj1 in the dorsal neural tube of mutants. (Y-Z) Quantification of dI interneuron subtypes in the dorsal neural tube at E10.5 and E11.5. Isl1-positive dI3 interneuron numbers are unchanged in Wnt1-Cre; Mib1 fl/fl embryos (Y), whereas AP2 α -positive dI2-5 interneuron numbers are increased in Wnt1-Cre; Mib1 fl/fl embryos at both E10.5 and E11.5 (Z). N=3-4 embryos; ns, not significant; *p=0.022; ***p=0.0001. Bar = 50μm.

Article Snippet: The following primary antibodies were used: mouse anti-Tuj1, 1:750 (Sigma-Aldrich T5076); mouse anti-Isl1, 1:50 (DSHB 40.3A4); rabbit anti-cleaved Caspase-3, 1:500 (Cell Signaling Technology 9661); mouse anti-AP2 α , 1:20 (DSHB 3B5); sheep anti-Dll1, 1:200 (R&D Systems AF5026); rabbit anti-N1ICD, 1:100 (Cell Signaling Technology 4147).

Techniques: In Situ Hybridization, Expressing, Immunostaining, Staining, Marker

Journal: bioRxiv

Article Title: Notch signaling is a critical initiator of roof plate formation as revealed by the use of RNA profiling of the dorsal neural tube

doi: 10.1101/2020.12.09.417279

Figure Lengend Snippet: (A-F) Transverse sections through the dorsal neural tube at the level of the forelimb in wildtype (WT) and Wnt1-Cre; Mib1 fl/fl mouse embryos, immunostained for AP2 α labeling neural crest and dorsal root ganglion (DRG) neurons (green), and cleaved-caspase 3 labeling apoptotic cells (red). At all stages examined, no cell death is evident in the premigratory neural crest or presumptive roof plate region. At E11.5 (E, F) increased cell death is evident in the DRG of Wnt1-Cre; Mib1 fl/fl embryos as previously reported (arrows). Bar = 100 μm. (G,H) Transverse sections at E10.5 immunostained for DLL1 (green) and EdU (red) following a 1hr EdU pulse to label proliferating cells. The dashed line indicates the boundary of DLL1 expression and defines the region used for quantification in (I). (I) Quantification of the number of EdU-positive proliferating cells in the Dll1-negative domain (presumptive roof plate) at E10.5, expressed as a percentage of the total number of DAPI-positive nuclei. Note the reduction in proliferating cells present in Wnt1-Cre; Mib1 fl/fl embryos compared to wildtype. N=6 embryos, **p=0.0018. Bar = 50μm.

Article Snippet: The following primary antibodies were used: mouse anti-Tuj1, 1:750 (Sigma-Aldrich T5076); mouse anti-Isl1, 1:50 (DSHB 40.3A4); rabbit anti-cleaved Caspase-3, 1:500 (Cell Signaling Technology 9661); mouse anti-AP2 α , 1:20 (DSHB 3B5); sheep anti-Dll1, 1:200 (R&D Systems AF5026); rabbit anti-N1ICD, 1:100 (Cell Signaling Technology 4147).

Techniques: Labeling, Expressing

Journal: Acta Pharmacologica Sinica

Article Title: Rifampicin induces clathrin-dependent endocytosis and ubiquitin–proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells

doi: 10.1038/s41401-019-0266-0

Figure Lengend Snippet: Primer sequences used in RT-qPCR

Article Snippet: A phospho (p)-PI3K polyclonal antibody (#4228), a PI3K monoclonal antibody (#4249), a p-p44/42 MAPK (ERK1/2) monoclonal antibody (#4370), a p44/42 MAPK (ERK1/2) monoclonal antibody (#4695), a p-SAPK/JNK antibody (#9251), a SAPK/JNK antibody (#9252), a p-P38 MAPK monoclonal antibody (#4511), a P38 MAPK antibody (#9212), a clathrin heavy-chain monoclonal antibody (#4796), a p-AP2 monoclonal antibody (#7399), a GAPDH monoclonal antibody (#2118), a ubiquitin antibody (#3933) and a Na + /K + ATPase antibody (#3010) were all obtained from Cell Signaling.

Techniques: Sequencing

Journal: Acta Pharmacologica Sinica

Article Title: Rifampicin induces clathrin-dependent endocytosis and ubiquitin–proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells

doi: 10.1038/s41401-019-0266-0

Figure Lengend Snippet: Rifampicin (RFP) affected the expression of clathrin and adaptor protein 2 (AP2) and the interactions of multidrug resistance-associated protein 2 (MRP2), clathrin and AP2 via oxidative stress. a The messenger RNA (mRNA) levels of clathrin and AP2 were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). b Western blot analysis was used to detect the expression of clathrin and the phosphorylation level of AP2. The relative protein expression levels of clathrin and p-AP2 were quantified with ImageJ. c Immunofluorescence staining for AP2 and clathrin (red). The nuclei were stained with Hoechst 33342 (blue). Scale bar, 5 μm. d Co-immunoprecipitation (Co-IP) was conducted to explore the interactions of MRP2, clathrin, and AP2. The values are expressed as the mean ± SD from three independent experiments ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. the control; & P < 0.05 vs. the RFP group. Magnification: ×400

Article Snippet: A phospho (p)-PI3K polyclonal antibody (#4228), a PI3K monoclonal antibody (#4249), a p-p44/42 MAPK (ERK1/2) monoclonal antibody (#4370), a p44/42 MAPK (ERK1/2) monoclonal antibody (#4695), a p-SAPK/JNK antibody (#9251), a SAPK/JNK antibody (#9252), a p-P38 MAPK monoclonal antibody (#4511), a P38 MAPK antibody (#9212), a clathrin heavy-chain monoclonal antibody (#4796), a p-AP2 monoclonal antibody (#7399), a GAPDH monoclonal antibody (#2118), a ubiquitin antibody (#3933) and a Na + /K + ATPase antibody (#3010) were all obtained from Cell Signaling.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Acta Pharmacologica Sinica

Article Title: Rifampicin induces clathrin-dependent endocytosis and ubiquitin–proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells

doi: 10.1038/s41401-019-0266-0

Figure Lengend Snippet: Rifampicin (RFP) caused clathrin-dependent endocytosis of multidrug resistance-associated protein 2 (MRP2). Western blot analysis was used to measure the transfection efficiency of small interfering RNAs (siRNAs) against clathrin and AP2 ( a – c ) and the expression of total MRP2 ( a , d ) and membrane MRP2 ( a , e ). f The membrane localization of MRP2 was determined with immunofluorescence (green). The nuclei were stained with Hoechst 33342 (blue). Scale bar, 5 μm. The values are expressed as the mean ± SD from three independent experiments ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001 indicate the results of pairwise comparisons of the siRNA groups. & P < 0.05 compared to the siRNA NC. # P < 0.05, ## P < 0.01, and ### P < 0.001 compared to the siRNA + RFP group. Magnification: ×400

Article Snippet: A phospho (p)-PI3K polyclonal antibody (#4228), a PI3K monoclonal antibody (#4249), a p-p44/42 MAPK (ERK1/2) monoclonal antibody (#4370), a p44/42 MAPK (ERK1/2) monoclonal antibody (#4695), a p-SAPK/JNK antibody (#9251), a SAPK/JNK antibody (#9252), a p-P38 MAPK monoclonal antibody (#4511), a P38 MAPK antibody (#9212), a clathrin heavy-chain monoclonal antibody (#4796), a p-AP2 monoclonal antibody (#7399), a GAPDH monoclonal antibody (#2118), a ubiquitin antibody (#3933) and a Na + /K + ATPase antibody (#3010) were all obtained from Cell Signaling.

Techniques: Western Blot, Transfection, Expressing, Immunofluorescence, Staining

Journal: Nucleic Acids Research

Article Title: Zfp217 mediates m 6 A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to promote adipogenic differentiation

doi: 10.1093/nar/gkz312

Figure Lengend Snippet: Zfp217 depletion impairs adipogenesis of 3T3L1 cells. ( A ) The mRNA and protein expression levels of Zfp217 were detected in 3T3L1 cells treated with siRNA for 36 h ( n = 3). ( B ) The adipogenic phenotypes of 3T3L1 cells transfected with si Zfp217 or siCtrl after MDI induction for 6 days were assessed by ORO staining; magnification: 200×. 3T3L1 cells were differentiated into adipocytes using MDI cocktail medium. ( C ) mRNA levels of adipogenic key genes PPARγ, aP2, LPL and Adiponectin at day 6 were detected by QPCR ( n = 3). ( D ) Protein levels of PPARγ and aP2 at day 6 were detected by western blot ( n = 3). ( E ) Schematic representation of knockout of Zfp217 . ( F ) The adipogenic phenotypes of WT and Zfp217 −/ - cells with the treatment of MDI for 6 days. ( G ) mRNA levels of PPARγ, aP2, LPL and Adiponectin from WT and Zfp217 −/ - cells with the treatment of MDI for 6 days ( n = 3). ( H ) Protein levels of PPARγ and aP2 from WT and Zfp217 −/ - cells with the treatment of MDI for 6 days ( n = 3). ( I ) Protein expression of Zfp217 in MEF-Zfp217 +/- cells. ( J ) The adipogenic phenotypes of MEF-Zfp217 +/- cells with the treatment of MDI for 6 days. ( K ) mRNA expression of adipogenic key genes in MEF-Zfp217 +/- cells with the treatment of MDI for 6 days ( n = 3). ( l ) protein expression of adipogenic key genes in MEF-Zfp217 +/- cells with the treatment of MDI for 6 days ( n = 3). Presented as means ± SD ( ** P < 0.01).

Article Snippet: The membranes were probed with the following antibodies against: Zfp217 (Abcam, USA, #48133), METTL3 (Abcam, #195352), ALKBH5 (Abcam, #69325), PPARγ (Cell Signaling Technology, USA, #C26H12), aP2 (Cell Signaling Technology, #D25B3), FTO (Santa cruz, USA, #sc-271713), YTHDF2 (Proteintech, China, #24744-1-AP), β-actin (Abclonal, China, #AC026), LMNB1 (Abclonal, #A1910) and Tublin (Abclonal, #AC021).

Techniques: Expressing, Transfection, Staining, Western Blot, Knock-Out

Journal: Nucleic Acids Research

Article Title: Zfp217 mediates m 6 A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to promote adipogenic differentiation

doi: 10.1093/nar/gkz312

Figure Lengend Snippet: Effect of Zfp217 deletion on the expression of target genes. ( A ) Validation of Ccdc141, Hspa1a and Efcab11 mRNA levels from RNA-seq data by QPCR ( n = 3). ( B ) Validation of m 6 A modification in Ccdc141, Hspa1a and Efcab11 mRNA by MeRIP-QPCR ( n = 3). ( C ) The adipogenic phenotypes of 3T3L1 cells transfected with si Ccdc141 , si Hspa1a , si Efcab11 or siCtrl after MDI induction for 6 days were assessed by ORO staining. ( D ) mRNA expression levels of adipogenic key genes PPARγ, aP2, LPL and Adiponectin were detected by QPCR ( n = 3). ( E ) Protein levels of PPARγ and aP2 were detected by western blot ( n = 3).Presented as means ± SD (* P < 0.05, ** P < 0.01).

Article Snippet: The membranes were probed with the following antibodies against: Zfp217 (Abcam, USA, #48133), METTL3 (Abcam, #195352), ALKBH5 (Abcam, #69325), PPARγ (Cell Signaling Technology, USA, #C26H12), aP2 (Cell Signaling Technology, #D25B3), FTO (Santa cruz, USA, #sc-271713), YTHDF2 (Proteintech, China, #24744-1-AP), β-actin (Abclonal, China, #AC026), LMNB1 (Abclonal, #A1910) and Tublin (Abclonal, #AC021).

Techniques: Expressing, RNA Sequencing Assay, Modification, Transfection, Staining, Western Blot

Journal: EBioMedicine

Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

doi: 10.1016/j.ebiom.2017.09.010

Figure Lengend Snippet: The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The results are representative of at least three independent experiments.

Article Snippet: Proteins were assessed with the following antibodies: anti-IRX3 antibody (Abcam, ab25703), anti-GFP antibody (Cell Signaling Technology, 2956s), anti-actin antibody (Santa Cruz Biotechnology, sc-8432), anti-UCP1 antibody (Alpha Diagnostic, ucp1-a), anti-HSP90 antibody (Cell Signaling Technology, 4877s), anti-PGC-1α antibody (Millipore, ab3242), anti-AP2 antibody (Cell Signaling Technology, 3544s), and horseradish peroxidase-conjugated (HRP)-linked secondary antibody (Cell Signaling Technology, 7076, 7074).

Techniques: Expressing, In Vitro, Isolation

Journal: Cell Death & Disease

Article Title: Epigenetic programming of Dnmt3a mediated by AP2 α is required for granting preadipocyte the ability to differentiate

doi: 10.1038/cddis.2016.378

Figure Lengend Snippet: Dnmt3a is upregulated by AP2 α during the CI stage. ( a ) Illustration of the murine Dnmt3a promoter (−1.7 to +0.1 kb) is shown and two reported AP2 α binding sites were identified in the proximal region. ( b ) Correlation coefficient analysis of AP2 α and Dnmt3a mRNA expression profiles during the CI stage. The mRNA expressions of AP2 α and Dnmt3a at different time points during the CI stage were determined by real-time PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase. ( c ) Real-time PCR analysis of the expression of Dnmt3a in AP2 α knockdown cells (si-1 and si-2) at ci48 h. The results were analyzed as means±S.D. ( n =3). Statistical significance is indicated: *** P <0.001. ( d ) Western blotting analysis of the expression of Dnmt3a in AP2 α knockdown cells at ci48 h. Cyclin-dependent kinase 4 served as the loading control. Quantification analysis of the relative protein level of Dnmt3a in AP2 α knockdown cells was shown on the right panel. The results were analyzed as means±S.D. ( n =3). Statistical significance is indicated: ** P <0.01. ( e ) A luciferase reporter plasmid of the murine Dnmt3a promoter (−1.7 to +0.1 kb) and an HA-AP2 α cDNA plasmid were constructed for dual-luciferase assay. Results were expressed as firefly luciferase activity normalized to renilla luciferase activity. Error bars indicate S.D. ( n =3). Statistical significance is indicated: * P <0.05, ** P <0.01. Gradient expression of HA-AP2 α was detected by western blotting (bottom panel)

Article Snippet: The following primary antibodies were used: anti-AP2 α (#3215; Cell signaling, Boston, MA, USA), anti-Dnmt3a (#2160; Cell signaling), anti-CDK4 (sc-260, Santa Cruz, CA, USA), anti-C/EBP β (sc-150; Santa Cruz), anti-C/EBP α (sc-61; Santa Cruz), anti-PPAR γ (sc-7273; Santa Cruz), anti-Lamin B (sc-6216; Santa Cruz), anti-rabbit IgG (sc-2027; Santa Cruz), anti-aP2 (AF1443; R & D Systems, Minneapolis, MN, USA), anti-Tubulin (T6199; Sigma, St. Louis, MO, USA), anti-HA (Tiangen, AB104) and anti -5-MeC (ab10805; Abcam, Cambridge, MA, USA).

Techniques: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Plasmid Preparation, Construct, Activity Assay

Journal: Cell Death & Disease

Article Title: Epigenetic programming of Dnmt3a mediated by AP2 α is required for granting preadipocyte the ability to differentiate

doi: 10.1038/cddis.2016.378

Figure Lengend Snippet: AP2 α transactivates the Dnmt3a promoter by directly binding to a small proximal promoter region. ( a ) Schematic diagram of promoter segments of the murine Dnmt3a gene (−1.7 to +0.1 kb) inserted in the pGL3 basic luciferase vector. ( b ) A series promoter segments of the Dnmt3a gene were co-transfected with pRL-TK (Renilla) in 3T3-L1 preadipocytes at ci0 h. Results were expressed as firefly luciferase activity normalized to renilla luciferase activity. Error bars indicate S.D. ( n =3). Statistical significance is indicated: *** P <0.001. ( c ) ChIP analysis of AP2 α binding to the target region on the Dnmt3a promoter (−0.6 to −0.4 kb). Chromatin samples of 3T3-L1 preadipocytes at ci48 h were subjected to ChIP assays by using an AP2 α antibody and normal rabbit IgG as a control. An upstream region (−1.1 to −0.9 kb) and a downstream region (−0.1 to +0.1 kb) in the Dnmt3a promoter were used as negative controls. ( d ) EMSA assay on the left panel (lane 1–12) showed the binding of endogenously expressed AP2 α to the target region on the Dnmt3a promoter under the condition of biotin-labeled DNA probes and cold competitors. The right panel (lane 13–16) showed the supershift (SS) results of endogenously expressed AP2 α in 3T3-L1 preadipocytes at ci48 h by anti-AP2 α antibody. Addition of anti-AP2 α antibody to the reaction resulted in the formation of the SS complex in lane 15. ( e ) Inactivation of the Dnmt3a promoter by mutating AP2 α binding site in the Dnmt3a promoter. 3T3-L1 preadipocytes at ci0 h were co-transfected with a luciferase reporter plasmid of the Dnmt3a promoter (wt or mut) and pRL-TK. Results were expressed as firefly luciferase activity normalized to renilla luciferase activity. Error bars indicate S.D. ( n =3). Statistical significance is indicated: *** P <0.001

Article Snippet: The following primary antibodies were used: anti-AP2 α (#3215; Cell signaling, Boston, MA, USA), anti-Dnmt3a (#2160; Cell signaling), anti-CDK4 (sc-260, Santa Cruz, CA, USA), anti-C/EBP β (sc-150; Santa Cruz), anti-C/EBP α (sc-61; Santa Cruz), anti-PPAR γ (sc-7273; Santa Cruz), anti-Lamin B (sc-6216; Santa Cruz), anti-rabbit IgG (sc-2027; Santa Cruz), anti-aP2 (AF1443; R & D Systems, Minneapolis, MN, USA), anti-Tubulin (T6199; Sigma, St. Louis, MO, USA), anti-HA (Tiangen, AB104) and anti -5-MeC (ab10805; Abcam, Cambridge, MA, USA).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Labeling

Journal: Cell Death & Disease

Article Title: Epigenetic programming of Dnmt3a mediated by AP2 α is required for granting preadipocyte the ability to differentiate

doi: 10.1038/cddis.2016.378

Figure Lengend Snippet: AP2 α knockdown impairs both genome-wide DNA methylation and the promoter methylation of adipogenic TFs during the CI stage. ( a ) 5-methylcytosine immunofluorescence of 3T3-L1 preadipocytes at ci48 h. The cells were transiently transfected with either scramble siRNA (ctrl) or AP2 α siRNAs (si-1 and si-2) at ci0 h and fixed at ci48 h for 5-methylcytosine immunofluorescence. Scale bars: 10 μ m. ( b ) Quantification analyses of total nuclear 5-methylcytosine densities in AP2 α knockdown cells at ci48 h. Data were collected at the same voltage and were presented as mean±S.D. ( n =3). Statistical significance is indicated: *** P <0.001. ( c ) DNA methylation status of C/EBPβ , C/EBPα , PPARγ and Egr2 promoters. 3T3-L1 preadipocytes were transiently transfected with either scramble siRNA (ctrl) or AP2 α siRNAs (si-1 and si-2) at ci0 h and collected at ci48 h. Genomic DNA samples were extracted and then subjected to bisulfite sequencing. Each column stands for a CpG site. The methylation percentage of each CpG site was shown by blue in each column. And the non-methylation percentage of each CpG site was shown by yellow in each column

Article Snippet: The following primary antibodies were used: anti-AP2 α (#3215; Cell signaling, Boston, MA, USA), anti-Dnmt3a (#2160; Cell signaling), anti-CDK4 (sc-260, Santa Cruz, CA, USA), anti-C/EBP β (sc-150; Santa Cruz), anti-C/EBP α (sc-61; Santa Cruz), anti-PPAR γ (sc-7273; Santa Cruz), anti-Lamin B (sc-6216; Santa Cruz), anti-rabbit IgG (sc-2027; Santa Cruz), anti-aP2 (AF1443; R & D Systems, Minneapolis, MN, USA), anti-Tubulin (T6199; Sigma, St. Louis, MO, USA), anti-HA (Tiangen, AB104) and anti -5-MeC (ab10805; Abcam, Cambridge, MA, USA).

Techniques: Genome Wide, DNA Methylation Assay, Methylation, Immunofluorescence, Transfection, Methylation Sequencing

Journal: Cell Death & Disease

Article Title: Epigenetic programming of Dnmt3a mediated by AP2 α is required for granting preadipocyte the ability to differentiate

doi: 10.1038/cddis.2016.378

Figure Lengend Snippet: Overexpression of Dnmt3a rescues the impairment of adipogenesis induced by AP2 α knockdown. (a) 3T3-L1 preadipocytes at ci0 h were transiently transfected with either scramble siRNA (ctrl) or AP2 α -specific siRNAs (si-1 and si-2), and then induced with hormone cocktail at ci48 h. On day 8, the cells were stained with Oil-Red-O (left panel), and then extracted with isopropanol and measured at OD 510 nm . The data were presented as means±S.D. ( n =3) and shown on the right panel. Statistical significance is indicated: * P <0.01. ( b ) Western blotting analysis of adipogenic factors on the cells induced by MDI for 8 days. ( c ) For rescue study, 3T3-L1 cells at ci0 h were transiently co-transfected with three pairs of plasmids (scramble siRNA (ctrl) and GFP plasmid, AP2 α -specific siRNA (si-1) and GFP plasmid, AP2 α -specific siRNA (si-1) and Dnmt3a plasmid), respectively. The cells were then induced with hormone cocktail after 48 h transfection. On day 8 of MDI induction, the cells were stained with Oil-Red-O (top panel) and extracted with isopropanol and measured at OD 510 nm (bottom panel). The data were presented as means±S.D. ( n =3). ( d ) Western blotting analysis of adipogenic factors on the cells induced by MDI for 8 days. ( e ) The expressions of Dnmt3a and AP2 α were analyzed with western blotting. Cyclin-dependent kinase 4 served as the loading control

Article Snippet: The following primary antibodies were used: anti-AP2 α (#3215; Cell signaling, Boston, MA, USA), anti-Dnmt3a (#2160; Cell signaling), anti-CDK4 (sc-260, Santa Cruz, CA, USA), anti-C/EBP β (sc-150; Santa Cruz), anti-C/EBP α (sc-61; Santa Cruz), anti-PPAR γ (sc-7273; Santa Cruz), anti-Lamin B (sc-6216; Santa Cruz), anti-rabbit IgG (sc-2027; Santa Cruz), anti-aP2 (AF1443; R & D Systems, Minneapolis, MN, USA), anti-Tubulin (T6199; Sigma, St. Louis, MO, USA), anti-HA (Tiangen, AB104) and anti -5-MeC (ab10805; Abcam, Cambridge, MA, USA).

Techniques: Over Expression, Transfection, Staining, Western Blot, Plasmid Preparation