Journal: Muscle & Nerve

Article Title: Leucine Supplementation Counteracts the Atrophic Effects of HDAC4 in Rat Skeletal Muscle Submitted to Hindlimb Immobilization

doi: 10.1002/mus.28411

Figure Lengend Snippet: Soleus muscle morphometric analysis after 7 days of hind limb immobilization and leucine supplementation in animals that overexpressed HDAC4. (A) Representative immunofluorescence photomicrographs of soleus muscle, laminin (green) and DAPI (blue, used for nuclei identification) (scale bar 100 μm) and hematoxylin–eosin staining (scale bar 50 μm). The asterisk corresponds to the same fiber in both HE and immunofluorescence technique. (B) Soleus muscle mass, (C) whole muscle CSA, (D) fiber CSA and (E) fiber distribution from EV, pCMVHDAC4, Imm/pCMVHDAC4 and Imm + Leu/pCMVHDAC4 groups. Data are expressed as mean ± SEM. Statistical analysis included the two‐way ANOVA test followed by Tukey's post hoc . * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 ( n = 5 per group).

Article Snippet: After 30 min, muscles were injected with 50 μL of the HDAC4 expression vector (1.0 μg/μL, pCMVHDAC4, Origene CAT#: RR217458, Rockville, MD, USA) or the empty vector (EV) and an electric pulse generator delivered a burst of pulses (6 pulses of 20 ms with 980 ms pause, 25 V).

Techniques: Immunofluorescence, Staining

Journal: Muscle & Nerve

Article Title: Leucine Supplementation Counteracts the Atrophic Effects of HDAC4 in Rat Skeletal Muscle Submitted to Hindlimb Immobilization

doi: 10.1002/mus.28411

Figure Lengend Snippet: HDAC4 protein expression analysis after 7 days of hind limb immobilization and leucine supplementation in animals that overexpressed HDAC4. (A) HDAC4 and GAPDH western blot showing representative bands. (B) Densitometry analysis by using GAPDH protein level as loading control. Data are expressed as mean ± SEM. Statistical analysis included the two‐way ANOVA test followed by Tukey's post hoc . ** p < 0.01 ( n = 5 per group).

Article Snippet: After 30 min, muscles were injected with 50 μL of the HDAC4 expression vector (1.0 μg/μL, pCMVHDAC4, Origene CAT#: RR217458, Rockville, MD, USA) or the empty vector (EV) and an electric pulse generator delivered a burst of pulses (6 pulses of 20 ms with 980 ms pause, 25 V).

Techniques: Expressing, Western Blot, Control

Journal: Muscle & Nerve

Article Title: Leucine Supplementation Counteracts the Atrophic Effects of HDAC4 in Rat Skeletal Muscle Submitted to Hindlimb Immobilization

doi: 10.1002/mus.28411

Figure Lengend Snippet: Effect of leucine on HDAC4 canonical pathway in animals that overexpressed HDAC4. (A) HDAC4 and (B) Atrogin‐1 mRNA levels were determined by qPCR. (C) Schematic illustrating HDAC4 action in the skeletal muscle fiber nucleus. Cyclophilin A expression was used as housekeeping. Data were expressed as mean ± SEM. Statistical analysis included one‐way ANOVA followed by Tukey's post hoc test. * p < 0.05; ** p < 0.01 ( n = 5 per group).

Article Snippet: After 30 min, muscles were injected with 50 μL of the HDAC4 expression vector (1.0 μg/μL, pCMVHDAC4, Origene CAT#: RR217458, Rockville, MD, USA) or the empty vector (EV) and an electric pulse generator delivered a burst of pulses (6 pulses of 20 ms with 980 ms pause, 25 V).

Techniques: Expressing

Journal: Muscle & Nerve

Article Title: Leucine Supplementation Counteracts the Atrophic Effects of HDAC4 in Rat Skeletal Muscle Submitted to Hindlimb Immobilization

doi: 10.1002/mus.28411

Figure Lengend Snippet: HDAC4 nuclei localization after 7 days of hind limb immobilization and leucine supplementation in animals that overexpressed HDAC4. (A) Representative immunofluorescence photomicrographs of HDAC4 in soleus muscle, HDAC4 (red), DAPI (blue, used for nuclei identification) (scale bar 100 μm). (B) Number of positive HDAC4 nuclei per total nuclei (%). Data were expressed as mean ± SEM. Statistical analysis included two‐way ANOVA followed by Tukey's post hoc test. * p < 0.05; ** p < 0.01 ( n = 5 per group).

Article Snippet: After 30 min, muscles were injected with 50 μL of the HDAC4 expression vector (1.0 μg/μL, pCMVHDAC4, Origene CAT#: RR217458, Rockville, MD, USA) or the empty vector (EV) and an electric pulse generator delivered a burst of pulses (6 pulses of 20 ms with 980 ms pause, 25 V).

Techniques: Immunofluorescence

Journal: International journal of biological sciences

Article Title: Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

doi: 10.7150/ijbs.69882

Figure Lengend Snippet: Figure 2. HDAC4 is responsible for deacetylation of GAC at K311. A, B Indicated plasmids were transfected into H1299 cells. Interaction between HDAC4 and GAC was detected by immunoprecipitation and western blot. WCL: whole cell lysate. C Indicated plasmids were transfected into H1299 cells. The protein expression was determined by western blot and glutaminase activity assay was performed. D Indicated siRNAs were transfected into H1299 cells. The protein expression was determined by western blot and glutaminase activity assay was performed. E Indicated plasmids and siRNAs were transfected into H1299 cells and glutaminase activity assay was performed. F The mitochondrial and cytosolic proteins in H1299 cells were separated and the location of HDAC4 and GAC was determined by western blot. VDAC was used as a marker of mitochondrial proteins and GAPDH was used as a marker of cytosolic proteins. Data are showed as mean ± SD, n=3. **P < 0.01, ns P >0.05.

Article Snippet: The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Activity Assay, Marker

Journal: International journal of biological sciences

Article Title: Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

doi: 10.7150/ijbs.69882

Figure Lengend Snippet: Figure 3. HDAC4 promoted cell proliferation and migration in NSCLC cells. A, B Indicated plasmids were transfected into H1299 (A) and A549 (B) cells and cell growth assay was performed. C, D Indicated siRNAs were transfected into H1299 (C) and A549 (D) cells and cell growth assay was performed. E, F Indicated siRNAs and plasmids were transfected into H1299 (E) and A549 (F) cells and cell growth assay was performed. Western blot assay was performed to confirm the transfection efficiency. G, H Indicated siRNAs were transfected into H1299 cells (G) and H292 cells (H) and cell wound healing assay was performed (scale bar: 500 µm, magnification: 100×). I F-actin staining assay. Indicated siRNAs were transfected into H1299 cells. 48 h later, cells were stained with phalloidin and DAPI. Scale bar=20 µm. Data are showed as mean ± SD, n=3. **P<0.01, ***P<0.001.

Article Snippet: The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene.

Techniques: Migration, Transfection, Growth Assay, Western Blot, Wound Healing Assay, Staining

Journal: International journal of biological sciences

Article Title: Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

doi: 10.7150/ijbs.69882

Figure Lengend Snippet: Figure 8. A working model of GAC acetylation in NSCLC. A working model depicting the molecular mechanism of HDAC4 mediated GAC deacetylation and TRIM21 mediated GAC ubiquitination to regulation tumorigenesis in NSCLC.

Article Snippet: The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene.

Techniques: Ubiquitin Proteomics

Journal: BMC Cancer

Article Title: Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4

doi: 10.1186/s12885-015-1561-x

Figure Lengend Snippet: HDAC4 is a target of miR-10b. a Ingenuity Pathway Analysis for targets of miR-10b. HDAC4 is shown with an arrow. b Sequence complementarities of miR-10b and its target HDAC4. c Luciferase assay was conducted to confirm that HDAC4 is a direct target of miR-10b. MCF-7 cells were co-transfected with dual luciferase plasmid pEZX-MT05-HDAC4-3′UTR along with a control pre-miR or pre-miR-10b, and assayed for luciferase activity 48 h after transfection. d Levels of HDAC4 in parental (MCF-7) and tamoxifen-resistance MCF-7 (MCF7TR) cells. e Effect of altered miR-10b levels on HDAC4 levels. β-actin protein was used as protein loading control for Western blots and RNU48 was used as internal control for the real-time RT-PCR miRNA analysis. C , control; PM , pre-miR-10b; AM , anti-miR-10b

Article Snippet: For luciferase reporter assays, MCF-7 cells were co-transfected with HDAC4 3′UTR luciferase vector (GeneCopoeia, Catalog # HmiT023167-MT05) and pre-miR-10b or miRNA negative control, using DharmaFECT Duo Transfection Reagent (Dharmacon).

Techniques: Sequencing, Luciferase, Transfection, Plasmid Preparation, Control, Activity Assay, Western Blot, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4

doi: 10.1186/s12885-015-1561-x

Figure Lengend Snippet: Effect of HDAC4 levels on tamoxifen-sensitivity. a siRNA against HDAC4 reduced its expression in MCF-7 cells. Functional role of HDAC4 and miR-10b on tamoxifen sensitivity in b MCF-7 and c MCF7TR cells. β-actin protein was used as protein loading control for Western blots. Tamoxifen treatment was done for 48 h at indicated doses. PM , pre-miR-10b; AM , anti-miR-10b; HDAC4 , HDAC cDNA; siHDAC4 , siRNA against HDAC4

Article Snippet: For luciferase reporter assays, MCF-7 cells were co-transfected with HDAC4 3′UTR luciferase vector (GeneCopoeia, Catalog # HmiT023167-MT05) and pre-miR-10b or miRNA negative control, using DharmaFECT Duo Transfection Reagent (Dharmacon).

Techniques: Expressing, Functional Assay, Control, Western Blot

Journal: BMC Cancer

Article Title: Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4

doi: 10.1186/s12885-015-1561-x

Figure Lengend Snippet: miR-10b and its target HDAC4 influence tamoxifen-induced apoptosis and cell viability. Effect of ectopic expression of miR-10b and HDAC4 on ( a ) apoptosis-induction and ( b ) viability of MCF-7 cells, and the effect of silencing of miR-10b and HDAC4 on ( c ) apoptosis-induction and ( d ) viability of MCF7TR cells. Tamoxifen treatment was for 48 h. PM , pre-miR-10b; AM , anti-miR-10b; HDAC4 , HDAC cDNA; siHDAC4 , siRNA against HDAC4

Article Snippet: For luciferase reporter assays, MCF-7 cells were co-transfected with HDAC4 3′UTR luciferase vector (GeneCopoeia, Catalog # HmiT023167-MT05) and pre-miR-10b or miRNA negative control, using DharmaFECT Duo Transfection Reagent (Dharmacon).

Techniques: Expressing