Journal: International Journal of Clinical and Experimental Pathology

Article Title: Formononetin sensitizes glioma cells to doxorubicin through preventing EMT via inhibition of histone deacetylase 5

doi:

Figure Lengend Snippet: Formononetin treatment decreased the expression of HDAC5. U87MG cells was treated with doxorubicin or in combination with formononetin (100 μM) for 48 h. Western blot was performed to determine the expression of HDAC5 in glioma cells incubated with doxorubicin (A and B) or in combination with formononetin (100 μM) (C and D). U87MG cells were transfected with the plasmid encoding HDAC5 (E) and cell viability was determined by CCK-8 methods after incubation with doxorubicin alone or combined with formononetin (F). *P < 0.05; **P < 0.01.

Article Snippet: The HDAC5 plasmid, HDAC5 siRNA and negative control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Incubation, Transfection, Plasmid Preparation, CCK-8 Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Formononetin sensitizes glioma cells to doxorubicin through preventing EMT via inhibition of histone deacetylase 5

doi:

Figure Lengend Snippet: Knockdown of HDAC5 diminished the doxorubicin-induced EMT in glioma cells. The HDAC5 siRNA-transfected glioma cells were incubated with doxorubicin alone or in combination with formononetin for 48 h. CCK-8 assay was used to determine the cell viability in different glioma cell lines including U87MG (A), U251MG (B) and T98G (C). Western blot analysis of E-cadherin and vimentin expression in HDAC5-siRNA or negative-siRNA transfected glioma cells (D and E). Relative protein expression of HDAC5 was quantified by band density with GAPDH served as control. *P < 0.05; **P < 0.01.

Article Snippet: The HDAC5 plasmid, HDAC5 siRNA and negative control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Transfection, Incubation, CCK-8 Assay, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Meteorin-like (Metrnl) adipomyokine improves glucose tolerance in type 2 diabetes via AMPK pathway

doi: 10.1101/420489

Figure Lengend Snippet: Metrnl increases GLUT4 expression by stimulating HDAC5 phosphorylation. a . Total mRNA from C2C12 cells were prepared after metrnl (100 ng/mL) treatment for the indicated times, and real-time qRT-PCR was performed using GLUT4-specific primers. For these assays, β-actin mRNA served as a positive control. b . C2C12 cells were stimulated with metrnl (100 ng/mL) for the indicate4d times. The cell lysates were analyzed by Western blotting using antibodies against GLUT4, with β-actin as a control. c . Time-dependent phosphorylation of HDAC5 after metrnl treatment. C2C12 cells were incubated with metrnl (100 ng/mL) for the indicated times. Cell lysates were analyzed by Western blotting usingantibodies against phospho-HDAC5 (Thr 498 ), while HDAC5 served as controls. d . C2C12 cells were pre-treated with compound C (30 μM), then treated with metrnl (100 ng/mL). Cell lysates were analyzed by Western blotting using an antibody against phospho-HDAC5 (Thr 498 ); HDAC5 served as a control. e . C2C12 cells were transiently transfected with AMPKα2 siRNA or non-target siRNA. Cell lysates were analyzed by Western blotting using antibodies against phospho-HDAC5 (Thr 498 ) and AMPKα2; HDAC5 and β-actin served as controls. f . C2C12 cells were treated with metrnl (100 ng/mL). Cytosolic and nuclear proteins were extracted from the cells. The phosphorylation of HDAC5 was evaluated by Western blot analysis. HDAC5 served as a control. Western blotting was performed on nuclear and cytosolic fractions to detect nuclear (lamin B) and cytosolic (α-tubulin) marker proteins. g . Representative images of phospho-HDAC5 treated with metrnl for 30?min. Scale bars, 10?μm. h . C2C12 cells were immunoprecipitated with anti-14-3-3 antibody, followed by Western blotting using anti-phospho-HDAC5. i . Representative images (phospho-HDAC5 and 14-3-3 objective images) of cells treated with metrnl for 1?h. Scale bars, 10?μm. j . The relative occupancy of HDAC5 and AcH3 on the GLUT4 promoter was assessed by qChIP analyses following 60 min of metrnl (100 ng/mL) treatment. The ChIP data represent IP values for each region’s ratio relative to the input. The results shown are from three independent experiments. * P < 0.05 and ** P < 0.01 vs. control, as indicated. Results are displayed as the mean ± SD from three experiments.

Article Snippet: HDAC5 was immunoprecipitated using an HDAC5 antibody (Novus Biologicals; Littleton, CO, USA), or an unrelated antibody (normal rabbit IgG) for a control.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Incubation, Transfection, Marker, Immunoprecipitation