Additional file 3: of An improved sequencing-based strategy to estimate locus-specific DNA methylation

Validation of the plasmid DNA standards. (PDF 277 kb

Additional file 6: of An improved sequencing-based strategy to estimate locus-specific DNA methylation

Inverse association between miR-200c/miR-141 locus methylation and miR-200c expression. (PDF 1483 kb

Additional file 5: of An improved sequencing-based strategy to estimate locus-specific DNA methylation

miR-200c/miR-141 locus methylation of MDA-MB-231 breast cancer cell line determined by BSP and NBSP performed with forward and reverse primers. (PDF 455 kb

HDAC4 and HDAC9 KO in SK-UT-1 cells.

<p>A) Immunoblot analysis of HDAC4, HDAC9 and MEF2D in the indicated SK-UT-1 clones. Two different HDAC9 KO clones were selected. Actin was used as loading control. B) mRNA expression levels of the indicated atypical and classical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC9. Data are presented as mean ± SD; n = 3. C) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC9. Dunn's Multiple Comparison Test was applied to test the significance. D) Immunoblot analysis of HDAC4 and HDAC9 in the indicated SK-UT-1 clones. Two different HDAC4 KO clones generated by different sgRNAs were selected. Actin was used as loading control. E) mRNA expression levels of the indicated atypical and classical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC4. Data are presented as mean ± SD; n = 3. F) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC4. Dunn's Multiple Comparison Test was applied to test the significance. G) Invasion properties of the SK-UT-1 cells WT, KO for HDAC4 or KO for HDAC9, as indicated. Data are presented as mean ± SD; n = 4. H) Example of growth in soft agar of SK-UT-1 cells WT or KO for HDAC9. Foci were stained with MTT. I) Quantitative results of colony formation assay for SK-UT-1 cells WT, KO for HDAC4 or KO for HDAC9, as indicated. Data are presented as mean ± SD; n ≥ 3. * p < 0.05, ** p < 0.01, *** p < 0.001</p

Epigenetic changes monitored by HDAC9.

<p>A) ChIP in SK-UT-1 cells WT or KO for HDAC9. Chromatin was immunoprecipitated using the anti-HDAC9 antibody. Normal rabbit IgGs were used as control. <i>TK</i> promoter was used as negative control. Atypical MEF2-targets are indicated in orange and the classical ones in blue. The genomic regions amplified by qPCR are the same as reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s009" target="_blank">S5E Fig</a>. Data are presented as mean ± SD; n = 4. B) ChIP in SK-UT-1 cells WT or KO for HDAC9. Chromatin was immunoprecipitated using the anti-H3K27ac and the H3K4me3 antibodies as indicated. Normal rabbit IgGs were used as control. <i>TK</i> promoter was used as negative control. Atypical MEF2-targets are indicated in orange and the classical ones in blue. The genomic regions amplified by qPCR are the same as reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s010" target="_blank">S6A and S6B Fig</a> (for H3H27ac) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s010" target="_blank">S6A and S6B Fig</a> (for H3K4me3). Data are presented as mean ± SD; n ≥ 4. C) Scatter dot plot representing the coding mRNA TPM levels of the four class IIa HDACs in a cohort of 26 LMS samples. The horizontal lines indicate the median. Dunn's Multiple Comparison Test was applied to test the significance of HDAC9 up-regulation. D) Scatter dot plot representing the IsoPCT of the two main isoforms of HDAC9: HDAC9 WT (red) and MITR (blue). Individual tumors (n = 26) were aligned along the x axis. E) Kaplan-Meier survival analysis related to the expression levels of HDAC9, performed on the same samples and according to the same criteria as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.g001" target="_blank">Fig 1H</a>. F) Summary of the shift in MEF2 transcriptional activities in relation to tumor progression in LMS. Possible co-activators and co-repressor are illustrated. The scheme describes the condition for the atypical MEF2-targets only. * p < 0.05, ** p < 0.01, *** p < 0.001.</p

MEF2D silencing causes opposite effect in SK-LMS-1 and SK-UT-1 cells.

<p>A) MEF2D expression was silenced by lentiviral infection using two different shRNAs. Immunoblot analysis of MEF2D and CDKN1A levels in SK-LMS-1 cells expressing the control shRNA or two different shRNAs against MEF2D. RACK1 was used as loading control. B) qRT-PCR analysis of mRNA expression levels of MEF2D and of some MEF2-target genes (<i>KLF2</i>, <i>RHOB</i>, <i>CDKN1A</i>, <i>JUN</i>, <i>CNN1</i>, <i>IRS1</i>) in SK-LMS-1 cells expressing the different shRNAs. mRNA levels are relative to control shRNA. Data are presented as mean ± SD; n = 4. C) Analysis of the cells synthetizing DNA as scored after BrdU staining. Mean ± SD; n = 3. D) SK-LMS-1 cells expressing the indicated shRNAs were seeded at 2x10⁴/ml in plates coated with 10μg/ml fibronectin, or BSA; after 16h they were subjected to time-lapse analysis for 6 hours. Results represent the individual migration rate and the average (bar) from at least 140 cells from three independent experiments. Mean and SEM are indicated. E) Invasion properties of the SK-LMS-1 cells expressing the shRNA2 against MEF2D or the control. Data are presented as mean ± SD; n = 4. Invasion of the Matrigel was scored after 16 hours and was expressed as ratio between cells invading the matrix in presence (oriented motility) and absence (random invasion) of the chemoattractant. Cells were evidenced with Hoechst 33342 staining. At least 5 fields for each condition were acquired and the invading cells were counted by using ImageJ. F) Growth in soft agar of SK-LMS-1 cells expressing the indicated shRNAs, foci were stained with MTT and counted. Data are presented as mean ± SD; n = 4. G) MEF2D expression was silenced by lentiviral infection using two different shRNAs. Immunoblot analysis of MEF2D and CDKN1A levels in SK-UT-1 cells expressing the control shRNA or two different shRNAs against MEF2D. RACK1 was used as loading control. H) qRT-PCR analysis of mRNA expression levels of MEF2D and of some MEF2-target genes (<i>KLF2</i>, <i>RHOB</i>, <i>CDKN1A</i>, <i>JUN</i>, <i>CNN1</i>, <i>IRS1</i>) in SK-UT-1 cells expressing the different shRNAs. mRNA levels are relative to control shRNA. Data are presented as mean ± SD; n = 4. I) Analysis of the cells synthetizing DNA as scored after BrdU staining. Mean ± SD; n = 3. J) SK-UT-1 cells expressing the indicated shRNAs were subjected to time-lapse analysis for 6 hours as in Fig 3D. Results represent the individual migration rate and the average (bar) from at least 140 cells from three independent experiments. Mean and SEM are indicated. K) Invasion properties of the SK-UT-1 cells expressing the shRNA2 against MEF2D or the control. Data are presented as mean ± SD; n = 4. Invasion of the Matrigel was scored after 16 hours and was expressed as ratio between cells invading the matrix in presence (oriented motility) and absence (random invasion) of the chemoattractant. Cells were evidenced with Hoechst 33342 staining. At least 5 fields for each condition were acquired and the invading cells were counted by using ImageJ. L) Growth in soft agar of SK-UT-1 cells expressing the indicated shRNAs, foci were stained with MTT and counted. Data are presented as mean ± SD; n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001</p

Analysis of MEF2D-HDAC4 repressive complexes in LMS cells.

<p>A) Quantitative analysis of the immunofluorescence studies. LMS cells were treated or not for 2 hours with 5ng/ml leptomycin B (LC Laboratories). After fixation of the cells, immunofluorescence analysis was performed to visualize HDAC4. Nuclei were stained with Hoechst 33342. Data are presented as mean ± SD (n = 3). B) MEF2D-HDAC4 complexes were immunoprecipitated using 1μg of anti-HDAC4, or anti-USP33, as a control, antibodies. Immunoblotting using an anti-MEF2D antibody was next used for the detection. The same amounts of cellular lysates were immunoprecipitated and the immunoblot were developed under the same circumstances. C) Chromatin was immunoprecipitated from SK-LMS-1 or SK-UT-1 cells using the anti-MEF2D and the anti-HDAC4 antibodies. Anti-FLAG antibody was used as control. <i>TK</i> promoter was used as negative control. The MEF2 binding site, the amplified region and the TSS are indicated for each tested gene, respectively with a vertical arrow, two arrowheads and a horizontal arrow. The TK promoter was used as negative control.</p

The MEF2 transcriptome.

<p>A) Venn diagrams showing the number of transcripts differing significantly in response to MEF2A and MEF2D silencing in SK-LMS-1 cells (green) or in SK-UT-1 cells (violet). Differentially expressed genes (DEGs) were selected based on fold change >1.5 and <-1.5 fold and p values <0.05. B) Gene ontology (GO) analysis was performed to interpret the principal biological processes under the regulation of MEF2 in SK-LMS-1 and SK-UT-1 cells. The NES (normalized enrichment score), the FDR (false discovery rate) and the p-value are provided. C) GSEA was performed by using SK-UT-1 DNA microarray data and genes repressed or induced by MEF2D/A KD in SK-LMS-1 cells, as indicated. Two groups were created in SK-UT-1 samples: A = shControl; B = shMEF2D/A. D) GSEA was performed by using SK-UT-1 DNA microarray data and genes repressed both by MEF2D/A KD in SK-LMS-1 and significantly modulated also in SK-UT-1 cells. Two groups were created in SK-UT-1 samples: A = shControl; B = shMEF2D/A. E) Scatter plot representing genes commonly down-regulated after MEF2A and MEF2D silencing in SK-LMS-1 cells (blue dots) and significantly modulated, after the same silencing, also in SK-UT-1 cells (red squares). F) Scatter plot representing genes commonly up-regulated after MEF2A and MEF2D silencing in SK-LMS-1 cells (blue dots) and significantly modulated, after the same silencing, also in SK-UT-1 cells (red squares). G) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-LMS-1 and SK-UT-1 cells in our DNA microarray and in another public available DNA microarray study (GSE39262). * p < 0.05, ** p < 0.01, *** p < 0.001</p

Defect in UPS-mediated MEF2-degradation in the most aggressive LMS cell line.

<p>A) Immunoblot analysis of HDAC4, SKP2 and MEF2 family members in LMS cells. Cells were treated for 8 hours with 2.5μM of the UPS inhibitor MG132. RACK1 was used as loading control. B) Immunoblot analysis of MEF2D isoforms in LMS cells engineered to express the dominant negative version of SKP2 (DN). RACK1 was used as loading control. C) Immunoblot analysis of MEF2D in LMS cells engineered to express an inducible version of SKP2 fused to ER as indicated. SKP2 was induced for 30 hours with 0.5μM 4-OHT. RACK1 was used as loading control and the nuclear relocalization of SKP2 after 4-OHT treatment was scored by immunofluorescence. D) Cellular lysates obtained in SK-LMS-1 cells expressing the DN mutant of SKP2 were immunoprecipitated using anti-MEF2Dα1 antibody and immunoblotted with the indicated antibodies. Immunoblots with total lysates (input) are also included. E) Cellular lysates obtained in SK-UT-1 cells expressing the DN mutant of SKP2 were immunoprecipitated using anti-MEF2Dα1 isoform and immunoblotted with the indicated antibodies. Immunoblots with total lysates (input) using the indicated antibodies are also included.</p

MEF2D supervised epigenetic changes on atypical and classical genes.

<p>A) Ratio between SK-LMS-1 and SK-UT-1 cells of H3K27ac, H3K4me3 and mRNA levels for a set of atypical and classical genes. <i>TK</i> was used as control. B) Ratio between SK-LMS-1 cells, WT and KD for MEF2D expression, of H3K27ac, H3K4me3 and mRNA levels for a set of atypical and classical genes. <i>TK</i> was used as control. C) Ratio between SK-UT-1 cells, WT and KD for MEF2D expression, of H3K27ac, H3K4me3 and mRNA levels for a set of atypical and classical genes. <i>TK</i> was used as control. Data are presented as mean ± SD; n ≥ 3. The binding of MEF2 was validated by ChIP (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s009" target="_blank">S5 Fig</a>) and the position of binding was expressed as relative to the major TSS, according to the hg38 assembly of the human genome. * p < 0.05, ** p < 0.01, *** p < 0.001</p