Cy3 dutp

Cells were cultured on slides. XIST probe and ATRX probe were respectively generated from XIST exon 1 DNA (GenBank U80460: 61251–69,449) and BACs including ATRX (RP11‐42 M11, BACPAC), which were labelled using nick translation kit (Roche) with Cy3‐dUTP (Amersham) and Cy5‐dUTP (Exon bio), respectively. RNA‐FISH was carried out as described previously.³⁸ We detected H3K27me3 (FITC 488) enrichment in cells with IF assays. Nuclear DNA was labelled by DAPI. Finally, slides were visualized with Leica TCS Sp8 confocal microscope (Zeiss) equipped with filters that are compatible for imaging with DAPI, Cy3, and Cy5.

To assess copy number and variations in X and Y chromosome structure, we performed DNA fluorescent in situ hybridization using BACs with known locations as DNA probes, as previously described.⁶³ (link) We labeled FISH probes by nick translation with biotin-dUTP (Sigma) and Cy3-dUTP (Amersham). To detect Biotin-labelled probes, we used Avidin-FITC probes (Life Technologies, 1:250 dilution), and counterstained DNA with DAPI. We captured images on a DeltaVision deconvolution microscope. We provide information on probes for FISH experiments in Table S7. For copy number analysis, we performed FISH on interphase cells using Whole X and Whole Y probes (Applied Spectral Imaging).

A FITC-labeled PNA probe (Panagene, Cat# F1009) was used in Terra RNA FISH. The amino-labeled oligonucleotide probe for detecting Terra was synthesized in-house. Nick translation labeled probes were prepared using nick translation kit (Roche, Cat# 10976776001). Nucleotide analogs used in probe labeling were Cy3-dUTP (Amersham, Cat# PA53022) and aminoallyl-dUTP (Jena Bioscience, Cat# NU-803S). Mouse Xist RNA was detected with Sx9 probe, a P1 DNA construct containing a 40 kb genomic fragment covering the mouse X inactivation centre (Xic) region. A ∼1 kb exon region of HOTAIR was PCR amplified from human genomic DNA using the primer pair: HOTAIR-F (5′-tgggagtgtgttttgttgga-3′) and HOTAIR-R (5′-gcacagaaaatgcatccaga-3′). A ∼1 kb exon region of NEAT2 was PCR amplified from human genomic DNA using the primer pair: MALAT1-F (5′-cttcctgtggcaggagagac-3′) and MALAT1-R (5′-gcacctgcagagaaaaggag-3′). The PCR amplicons were cloned into plasmid vectors. Purified plasmid DNA was used as template DNA in nick translation to generate probes targeting the corresponding lncRNAs. Chromosome X paint was purchased from Cambio (Cat# 1189-XMF-02).

RNA FISH, immunostaining and immuno-RNA FISH were carried out as previously described (16 (link)). Immunostaining for H3K27me3 was performed using a mouse monoclonal antibody (Abcam; ab6002; 1:500) with a secondary antibody conjugated with Alexa-647 (ThermoFisher; A-21236; 1:1000). Immunostaining for H2AK119ub was performed using a rabbit monoclonal antibody (Cell Signaling Technology; D27C4; 1:2000) with a secondary antibody conjugated with Alexa-647 (Abcam; ab150075; 1:1000). Immunostaining was followed by RNA FISH. The Xist RNA was detected with Sx9 probe, a P1 DNA construct containing a 40 kb genomic fragment covering the Xist gene. The Usp9x probe was prepared with a BAC DNA (RP24-306P3). Nucleotide analogs used in probe labeling were Cy3-dUTP (Amersham, Cat# PA53022).

MSCs were incubated for 3 d with icariin (1 µM) in the AS or OS medium, respectively. Total RNA was extracted and reverse-transcripted to cDNA with SuperScript III reverse transcriptase (Invitrogen). Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ) was incorporated during reverse transcription of 40 μg of purified total RNA. Different fluorescent-labeled cDNA samples were mixed and competitively hybridized to a homemade cDNA microarray [26 (link)], which contained 1000 genes selected from a mouse clone set. Microarrays were scanned using a microarray scanner (ScanArray 4000, GSI Lumonics, MA), and images were analyzed with GenePix Pro 4.0 software (Axon, CA). Genes showing greater than 2-fold induction or repression (Cy5/Cy3 ratios > 2 or <0.5) were selected for further analysis. The Protein Analysis Through Evolutionary Relationships (PANTHER) classification system was applied to analyze the functional classification and correlated pathways (http://www.pantherdb.org) [27 (link)].

To assess copy number and variations in X and Y chromosome structure, we performed DNA fluorescent in situ hybridization using BACs with known locations as DNA probes, as previously described.^{57 (link)} We labelled FISH probes by nick translation with biotin-dUTP (Sigma) and Cy3-dUTP (Amersham). To detect Biotin-labelled probes, we used Avidin-FITC probes (Life Technologies, 1:250 dilution), and counterstained DNA with DAPI. We captured images on a DeltaVision deconvolution microscope. We provide information on probes for FISH experiments in Table S7. For copy number analysis, we performed FISH on interphase cells using Whole X and Whole Y probes (Applied Spectral Imaging).