Cavalieri V , Spinelli G .

	Fig 1. Effect of HDAC inhibition on the expression of the hbox12 gene.(A) P. lividus embryos cultured in the absence or in the presence of either TSA or VPA at the indicated dosages, and observed at the early blastula stage. (B) Northern blot analysis of total RNA isolated from embryos at the early blastula stage treated or not treated with TSA or VPA, and probed with an antisense 32P labelled RNA against the hbox12 transcript. The lower panel shows the loading control ribosomal RNAs in the ethidium bromide stained agarose gel. (C) qPCR measurements of hbox12 transcript abundance in blastulae treated with 50 nM TSA. Data are shown as normalized ΔCt (ΔΔCt, left ordinate), and as the corresponding fold difference in transcript abundance (right ordinate), with respect to control unperturbed embryos at the same stages of development. The gray region represents ΔΔCt values corresponding to less than 3-fold difference. Error bars are standard errors for the qPCR replicates. Oligonucleotide primer pairs used for qPCR reactions and amplicon lengths are indicated in the S1 Table. (D) Spatial distribution of the hbox12 transcripts in control and TSA-treated embryos at the early blastula stage, revealed by chromogenic WMISH.
	Fig 2. Impact of TSA on acetylation of H3K9 and hbox12 promoter activity.(A) Western blot analysis using the anti-H3K9ac antibody. Nuclear extracts from control and 50 nM TSA-treated blastula stage embryos were fractioned by SDS-PAGE (on the left is shown the gel staining), blotted on PVDF membrane and incubated with anti-H3K9ac antibody. (B and C) ChIP-qPCR analysis of the hbox12 promoter occupancy by H3K9ac and HDAC-1. ChIP assays were performed on chromatin extracted from control and TSA-treated embryos at the mesenchyme blastula stage and precipitated with commercial antiserum against H3K9ac or HDAC-1, or incubated without adding antibodies, followed by qPCR amplification of an hbox12 promoter fragment. Data are normalized according to the percent of input method. Bars are as in Fig 1C. (D) Zygotes were injected with the phbox12-GFP transgene, the resulting embryos were raised in the presence of TSA 50 nM, and observed at the indicated stages. Images for each embryo are shown under DIC optic superimposed on epifluorescence field. (E) qPCR measurements of gfp transcript abundance in blastulae treated with 50 nM TSA. Data are normalized and indicated as in Fig 1C.
	Fig 3. TSA treatment and effect on nodal gene expression.(A) Northern blot analysis of total RNA isolated from embryos at the indicated stages exposed or not to TSA 50 nM, and probed with an antisense 32P labelled pRNA against the nodal transcript. The lower panel shows the loading control ribosomal RNAs in the ethidium bromide stained agarose gel. (B) Control and TSA-treated embryos were fixed at the early blastula stage, and analysed by WMISH with a DIG-labelled nodal probe. (C) ChIP-qPCR analysis of the nodal promoter occupancy by H3K9ac. As a negative control, chromatin samples were incubated without antibodies, and negligible amplification was obtained from the corresponding purified DNA. Data are normalized according to the percent of input method, and shown as in Fig 2B and 2C.
	Fig 4. Rescue of nodal transcription by HD expression into TSA-treated embryos.(A) 0.1–0.2 pg of the hd mRNA, or a control out-of-frame strim1 transcript [53], were injected into zygotes exposed to TSA and total RNA isolated from the resulting embryos at the early blastula stage. (B) qPCR measurements of relative nodal transcript abundance in embryos exposed to TSA and injected with increasing amounts of the hd mRNA, compared to the nodal mRNA level of control unperturbed embryos. Data are normalized and indicated as in Fig 1C. (C) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd mRNA together with the TRCD red fluorescent tracer, and the phenotype of the resulting embryos was examined at the gastrula stage. (D) Representative examples of control gastrulae, TSA-treated larvae, and rescued embryos at the same stage injected with the hd mRNA, respectively ordered from left to right. Note that in both the rescued embryos, the progeny of the blastomere that received hd was embedded into the ventral side. (E) At the 4-cell stage, one blastomere of TSA-treated embryos was injected with the hd-gfp mRNA, and the resulting embryos at the early blastula stage were disaggregated into individual cells that were eventually segregated into two populations, based on GFP fluorescence, by way of FACS. (F) Representative example of embryos expressing HD-GFP clonally, observed at the early blastula stage, just before dissociation. (G) Changes in gene expression level of nodal assessed by qPCR in whole embryos exposed to TSA, and in subpopulations of cells sorted from rescued TSA-treated embryos clonally expressing HD-GFP. Data are normalized and indicated as in Fig 1C.

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