Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: RUNX1T1-coding sequence (RUNX1T1-CDS) primers were designed for amplifying the open reading frame (ORF) of RUNX1T1 ( a ) and the sketch of RUNX1T amplification product from Hu lamb ( b ). The four lanes in the red bracket represented RUNX1T1 amplification product using subcutaneous fat cDNA ( a ). The dashed line represents the 5′ untranslated region of RUNX1T1 ( b ) .

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Sequencing, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: The cDNA fragment and predicted protein sequences of ovine RUNX1T1. The start and stop codons are framed in red. The protein sequence was predicted by the BLAST tool of DNAMAN (version 6.0, LynnonBiosoft, San Ramon, CA, USA) and is presented under the coding sequence. All sequences were aligned by the DNAMAN (version 6.0, LynnonBiosoft) software. * means the translation is terminated.

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Sequencing, Software

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Comparison of the bovine, mouse, and human RUNX1T1 amino acid sequences. The RUNX1T1 amino acid sequence (Accession No.: MH063277) was aligned to those of bovine (Accession No.: NP_001092855), murine (Accession No.: EDL05616), and human (Accession No.: NP_001185608.1) by DNAMAN. The same amino acid residues in four, three and two species are highlighted in red, green, yellow respectively, while the amino acid residues present in only one species are highlighted in white. The apostrophes indicates that these amino acid sequences are absent.

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Comparison, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Expression pattern of RUNX1T1 isoforms in different tissues from 6-month-old Hu sheep. ( a ) Primer I was designed for identifying RUNX1T1 isoforms, primer II was used for qPCR of RUNX1T1-L mRNA. The red line represents the region which is present in the long isoform and absent in the short isoform. Depending on isoforms, bands of either 456 bp or 211 bp can be detected ( b ). Real-time PCR of mRNA for RUNX1T1-L (178 bp; ( c )) and FTO ( f ) in different tissues. Expression of the RUNX1T1-S (211 bp) in different tissues were determined by semi-qRT-PCR ( d , e ). Expression of gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), and relative to the expression in brain. Quantitative data are represented as the mean ± SEM ( n = 5).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Expression patterns of RUNX1T1 isoforms in the subcutaneous fat. The temporal expression of RUNX1T1 isoforms ( a – c ) and FTO mRNA ( d ) and RUNX1T1 protein ( e ) in the subcutaneous fat of Hu sheep at different ages were measured using Real-time PCR and Western blot (5 d, 3 M, 6 M and 24 M). d means day, M means month. Semi-quantitative analysis of the relative RUNX1T1 protein expression levels in the subcutaneous fat at different developmental stages was undertaken ( f ). Real-time PCR was performed on different developmental stages of subcutaneous fat to determine the expression level of marker genes ( g ), and immunofluorescence localization of RUNX1T1 in the subcutaneous fat of 24-month-old Hu sheep ( h – j ). Blue color indicates 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei ( h ). Red color indicates the expression of RUNX1T1 ( i ). Pictures in ( g ) and ( h ) were merged ( j ). Scale bars = 100 µm. One-way ANOVA, and bars with different letters are significantly different ( p < 0.05). Quantitative data are shown as mean ± SEM ( n = 5).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Expression patterns of RUNX1T1 isoforms during ovine preadipocyte differentiation. The temporal expression pattern of RUNX1T1 isoforms and FTO during ovine preadipocyte differentiation ( a , b ); image of newly isolated and adipogenic differentiated ovine preadipocytes ( c ). Scale bars = 100 µm. One-way ANOVA followed by comparison of all time points to day 0 with Tuckey post hoc analysis, * p < 0.05. Quantitative data are represented as the mean ± SEM ( n = 4).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Expressing, Isolation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Adipogenic differentiation of ovine preadipocytes with RUNX1T1-L knockdown. siRUNX1T1-L and siNC were introduced into ovine preadipocytes when the density reached about 70%, and 48 h later, cells were subject to adipogenic differentiation ( a ). The expression of RUNX1T1-L was determined at 48 h post-transfection by Real-time PCR. Moreover, the expression of RUNX1T1-S was analyzed by semi-qRT-PCR ( b ). Ovine preadpocytes were transfected with siRUNX1T1-L or siNC and differentiated into mature adipocytes ( c ). Scale bars = 100 µm. After 8 days of differentiation, intracellular lipid droplets were stained using Oil Red-O. Lipid content was indirectly determined by measuring the optical density (OD) value at 510 nm on a spectrophotometer ( d ). Real-time PCR was performed on day 6 of adipogenic differentiation to determine the mRNA level of maker genes ( e ). Independent Students’ t -test. * p < 0.05 against siNC. Quantitative data are represented as the mean ± SEM ( n = 4).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Knockdown, Expressing, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Spectrophotometry

Journal: MedComm

Article Title: Inhibition of FOS‐Like Antigen 1 Reduces Chemoresistance to Temozolomide Through Stemness Reprogramming via IL‐6/STAT3 Tyr705 Pathway

doi: 10.1002/mco2.70593

Figure Lengend Snippet: Inhibition of FOSL1 in GBM cells reverses TMZ response. (A, B) Representative histogram of protein expression, including FOSL1 and cell cycle‐associated molecules, in GBM patient's cells was shown (top panel). Protein expression in patients with GBM‐derived cells ( n = 6), including FOSL1 and cell cycle‐associated molecules, was analyzed by flow cytometry and quantified using FlowJo V10 (bottom panel). (C) FOSL1 mRNA expression (left panel) in U87MG treated with si‐control or si‐FOSL1 analyzed using qPCR. GAPDH was used as a control gene for relative quantification. FOSL1 protein expression in U87MG treated with si‐control or si‐FOSL1 was analyzed by flow cytometry and quantified using FlowJo V10 (right panel). (D) Expression of G0/G1 to S phase transition‐related proteins, including CDK4, cyclin D, CDK2, and cyclin E, analyzed using flow cytometry and quantified using FlowJo V10. E Population of the G0/G1 phase of U87MG cells stained with CCS1 and analyzed using flow cytometry. Data were quantified using FlowJo V10. (F) Representative images of colony formation showing viable U87MG cells treated with si‐control, si‐FOSL1 or TMZ (purple). (G) Proliferation of U87MG cells calculated using the WST‐8 reduction assay following the manufacturer's instructions. *p < 0.05; **p < 0.005; ***p < 0.0005; paired t ‐test ( n = 3).

Article Snippet: For intracellular protein analysis, U87MG cells and patient‐derived GBM cells were collected and incubated in permeabilization buffer (#554722; BD Biosciences) at 4°C for 20 min. After washing with Perm/Wash Buffer, cells were stained at 4°C for 30 min using the following primary antibodies: anti‐FOSL1 (#PA5‐76185; Thermo Fisher Scientific), CDK2 (#LS‐C351983; LSBio), CDK4 (#LS‐ C99873 ; LSBio), cyclin D (#LS‐B4507; LSBio), cyclin E (#32‐1600; Thermo Fisher Scientific), Oct4 (#ab184665; Abcam), Nanog (#ab109250; Abcam), Sox2 (#ab171380; Abcam), MGMT (#MA5‐13506; Thermo Fisher Scientific), IL‐6 (#M620; Thermo Fisher Scientific), STAT3 (#710077; Thermo Fisher Scientific), phospho‐STAT3 Ser727 (#PA5‐17876; Thermo Fisher Scientific), phospho‐STAT3 Tyr705 (#MA5‐15193; Thermo Fisher Scientific), and intracellular ROS (#ab113851; Abcam).

Techniques: Inhibition, Expressing, Derivative Assay, Flow Cytometry, Control, Quantitative Proteomics, Sublimation, Staining