6 research outputs found
CTS-induced HDAC4 nuclear relocation in chondrocytes.
<p>(A) Fluorescence microscope showed that GFP-HDAC4 was mainly located in the cytoplasm of cells in non-stretched control group (a-c), while GFP-HDAC4 was relocated to nucleus of cells subjected to CTS (d-f). Green indicated the GFP-HDAC4 and blue indicated cell nuclei stained by Hoechst 33342. (B) Percentage of GFP-HDAC4 located in nucleus was scored. 300 cells from 3 independent experiments were counted. Data were expressed as meansĀ±SD (P = 0.006). (C) Nuclear and cytoplasmic lysates were separated and followed by western blot analysis with anti-HDAC4 antibody. Histone 3 and GAPDH acted as loading controls for the nuclear and cytoplasmic fraction respectively (C-a). Semi-quantitative assay of band densities showed that cytoplasmic HDAC4 was decreased, and nuclear HDAC4 was increased in CTS group as compared to non-stretched control group (C-b), Values were presented as meanĀ±SD (n = 3).</p
OA impairs the HDAC4 nuclear import induced by CTS.
<p>(A) Fluorescence microscope showed GFP-HDAC4 was mainly located in the nucleus in cells subjected to CTS only (a-c), and mainly in the cytoplasm in cells subjected to CTS with OA (d-f). Green indicated the GFP-HDAC4, and blue indicated cell nucleus stained by Hoechst 33342. (B) Percentage of green GFP-HDAC4 located only in nucleus was scored. 300 cells from 3 independent experiments were counted. Data were expressed as meansĀ±SD (P = 0.02). (C) Nuclear and cytoplasmic lysates were separated and followed by western blot analysis with anti-HDAC4 antibody. Histone 3 and GAPDH acted as loading controls for the nuclear and cytoplasmic fraction respectively (C-a). Semi-quantitative assay of band densities showed that cytoplasmic HDAC4 was decreased, and nuclear HDAC4 was increased in CTS group as compared to control group and CTS with OA group; The relative grey value of HDAC4 had no statistics difference between control group and CTS with OA group in both cytoplasm and nucleus (C-b). Values were presented as meanĀ±SD (n = 3), *P<0.05.</p
Overview of experimental design.
<p>(A) Workflow scheme for analysis of the effect of cyclic equibiaxial tensile strain (CTS) on HDAC4 relocation in chondrocytes. After isolated and then cultured for 6 days, the passage chondrocytes were transfected with GFP-HDAC4. At 48 h post-transfection, the transfected cells were submitted to CTS for 3 hours. (B) The chondrocytes at Area A were subjected to 6% equibiaxial CTS. (C) Green fluorescent protein (GFP) was captured by fluorescence microscope to validate the transfection efficiency of GFP-HDAC4 in chondrocytes. Nuclei were stained by Hoechst 33342 (blue) (C-a). 300 cells from 3 independent experiments were counted. Transfection efficiency of GFP-HDAC4 is 96%Ā±3.64% (C-b). (D, E) The time-dependent changes in gene expression levels for aggrecan and type II collagen following CTS. Real-time PCR showed that mRNA level for aggrecan (D) and collagen II (E) were elevated after 2 and 3 h of CTS, but decreased after 4 h of CTS. Values are presented as meanĀ±SD (n = 3). *P<0.05, **P<0.01 versus the non-stretched control group.</p
A model of HDAC4 cytoplasmic-nuclear relocation involved in gene expression in response to CTS.
<p>CTS induced HDAC4 relocation from cytoplasm to the nucleus. When HDAC4 relocated to the nucleus, it increases chondrogenic gene expression and reduces hypertrophic gene expression in the chondrocytes.</p
Inhibition of HDAC4-nuclear-import by OA abrogates the CTS-induced change of gene expression.
<p>(A) Real-time PCR was carried out to analyze the mRNA levels for aggrecan (a), collagen II (b), LK1 (c), SOX9 (d), Runx2 (e), Ihh (f), collagen X (g) and MMP-13 (h) in non-stretched without OA control group, CTS group and CTS with OA group. Values were presented as meanĀ±SD (n = 3). *P<0.05. (B) OA did not induce cell death. The viability of cells subjected to CTS with OA was assessed by using Hoechst 33342/PI double staining at 48 h post-CTS. The cells frozen at -20Ā°C served as positive controls (B-a to c). No dead cells were detected in CTS with OA groups (B-d to f). Blue indicated cell nucleus stained by Hoechst 33342, while red indicated PI stain in dead cells.</p
Transcriptome Analysis of <i>Barbarea vulgaris</i> Infested with Diamondback Moth (<i>Plutella xylostella</i>) Larvae
<div><p>Background</p><p>The diamondback moth (DBM, <i>Plutella xylostella</i>) is a crucifer-specific pest that causes significant crop losses worldwide. <i>Barbarea vulgaris</i> (Brassicaceae) can resist DBM and other herbivorous insects by producing feeding-deterrent triterpenoid saponins. Plant breeders have long aimed to transfer this insect resistance to other crops. However, a lack of knowledge on the biosynthetic pathways and regulatory networks of these insecticidal saponins has hindered their practical application. A pyrosequencing-based transcriptome analysis of <i>B. vulgaris</i> during DBM larval feeding was performed to identify genes and gene networks responsible for saponin biosynthesis and its regulation at the genome level.</p><p>Principal Findings</p><p>Approximately 1.22, 1.19, 1.16, 1.23, 1.16, 1.20, and 2.39 giga base pairs of clean nucleotides were generated from <i>B. vulgaris</i> transcriptomes sampled 1, 4, 8, 12, 24, and 48 h after onset of <i>P. xylostella</i> feeding and from non-inoculated controls, respectively. <i>De novo</i> assembly using all data of the seven transcriptomes generated 39,531 unigenes. A total of 37,780 (95.57%) unigenes were annotated, 14,399 of which were assigned to one or more gene ontology terms and 19,620 of which were assigned to 126 known pathways. Expression profiles revealed 2,016ā4,685 up-regulated and 557ā5188 down-regulated transcripts. Secondary metabolic pathways, such as those of terpenoids, glucosinolates, and phenylpropanoids, and its related regulators were elevated. Candidate genes for the triterpene saponin pathway were found in the transcriptome. Orthological analysis of the transcriptome with four other crucifer transcriptomes identified 592 <i>B. vulgaris</i>-specific gene families with a <i>P</i>-value cutoff of 1e<sup>ā5</sup>.</p><p>Conclusion</p><p>This study presents the first comprehensive transcriptome analysis of <i>B. vulgaris</i> subjected to a series of DBM feedings. The biosynthetic and regulatory pathways of triterpenoid saponins and other DBM deterrent metabolites in this plant were classified. The results of this study will provide useful data for future investigations on pest-resistance phytochemistry and plant breeding.</p></div