Gene network analysis of differentially expressed transcripts between mESC-derived Hb9 control and A2 SMA MNs.

<p>Ingenuity Pathways Analysis (IPA) of biological functions for transcripts (<b>A</b>) upregulated or (<b>B</b>) downregulated in A2 SMA MNs compared to Hb9 control MNs. IPA of canonical pathways for transcripts (<b>C</b>) upregulated or (<b>D</b>) downregulated in A2 SMA MNs compared to Hb9 control MNs. IPA was performed on upregulated or downregulated transcripts with at least a 2-fold change and a p value less than or equal to 0.05. The top 7 canonical pathways or biological functions were shown in the bar graphs. The top canonical pathway for (<b>E</b>) upregulated (Transcriptional Networks in ESCs) or (<b>F</b>) downregulated (Notch Signaling) transcripts in A2 SMA MNs compared to Hb9 control MNs. Downregulated transcripts are green while the upregulated transcripts are red.</p

Validation of differentially expressed transcripts determined by RNA-Seq using qRT-PCR.

<p>The levels of <i>Crabp1</i>, <i>Crabp2</i>, <i>Isl1</i>, <i>Nkx2.2</i>, <i>Pla2g1b</i>, <i>Smn1</i> and <i>Vim</i> transcripts were measured in total RNAs from control and SMA mESC-derived MNs. (<b>A</b>) The magnitude of change (log₂(fold change)) of selected transcripts in A2 SMA MNs relative to Hb9 control MNs (n = 3/genotype) as determined by RNA-Seq (black bars) or qRT-PCR (grey bars). (<b>B</b>) The magnitude of change of selected transcripts in HB9:eGFP-labeled (black bars) or unlabeled (grey bars) SMA MNs (A2 and E2 cells, respectively) relative to control MNs (Hb9 and C4 cells, respectively; n = 3/genotype).</p

Upstream regulator analysis of differentially expressed transcripts between mESC-derived Hb9 control and A2 SMA MNs.

<p>IPA of significantly modified upstream (<b>A</b>) transcriptional regulator, (<b>B</b>) endogenous signaling and (<b>C</b>) drug pathways in upregulated or downregulated transcripts in A2 SMA MNs compared to Hb9 control MNs. Significant upstream regulators were identified as those having an activation z-score greater than or equal to 2.0 for activated regulators or less than or equal to −2.0 for inhibited regulators.</p

RNA-Seq reads mapped to NCBI mouse genome build 37 using TopHat.

<p>RNA-Seq reads mapped to NCBI mouse genome build 37 using TopHat.</p

RNA-Seq-identified differentially expressed transcripts between mESC-derived Hb9 control and A2 SMA MNs.

<p>(<b>A</b>) Flowchart outlining the analysis of RNA sequencing data. The programs used for each step are shown to the right of the flowchart. (<b>B</b>) Area plot showing the distributions of FPKM values of significant transcripts in Hb9 control (blue) and A2 SMA (orange) MNs. (<b>C</b>) A volcano plot showing the log2-fold difference of statistically significant (p = 0.05) transcripts (shown in blue) between Hb9 control and A2 SMA MNs.</p

Expression of RNA-Seq-identified differentially expressed transcripts in SMA mouse spinal cords.

<p>The levels of <i>Crabp1</i>, <i>Crabp2</i>, <i>Isl1</i>, <i>Nkx2.2</i>, <i>Pla2g1b</i>, <i>Smn1</i> and <i>Vim</i> transcripts were measured in spinal cord total RNAs from control and SMA mice. (<b>A</b>) The magnitude of change (log₂(fold change)) of selected transcripts in low copy <i>SMN2</i> SMA (<i>SMN2(89)^+/+;mSmn^−/−</i>; black bars) or high copy <i>SMN2</i> rescue (<i>SMN2(566)^+/+;mSmn^−/−</i>; grey bars) mouse spinal cord samples (n = 3/genotype) relative to control samples. (<b>B</b>) The magnitude of change of selected transcripts in low copy SMN2 SMA mouse spinal cord samples relative to controls at embryonic day 13.5 (e13.5; black bars) and postnatal day 3 (PND03; grey bars).</p

Relationship between changes in mRNA levels measured by RNA-Seq and protein levels measured by 2D-DIGE or immunoblot (*) of selected genes in normal versus SMA mESC-derived motor neurons.

<p>The protein expression data is taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106818#pone.0106818-Wu1" target="_blank">[46]</a>.</p><p>Relationship between changes in mRNA levels measured by RNA-Seq and protein levels measured by 2D-DIGE or immunoblot (*) of selected genes in normal versus SMA mESC-derived motor neurons.</p

Sequences of primers used for qRT-PCR.

<p>Sequences of primers used for qRT-PCR.</p

Expression of total SMN protein in undifferentiated as well as MN-differentiated control and SMA cells.

<p>Representative immunoblots of both differentiated and undifferentiated mESCs revealed a significantly reduced protein expression of SMN in A2 SMA cell lines compared to Hb9 control cells. SMN levels were normalized to Gapdh. C =  control, S =  SMA. Asterisks indicate significant differences compared to controls (*p<0.05, Student's <i>t</i> test).</p

Functionally regulated gene networks in mESC-derived A2 SMA and Hb9 control MNs.

<p>The highest scoring interactomes generated by IPA from the (A) upregulated or (<b>B</b>) downregulated transcripts in A2 SMA MNs when compared against Hb9 control MNs. Each node represents a gene product in the network. Upregulated gene products are shown in red while the downregulated gene products are shown in green; the intensity of color indicates the degree of regulation. The uncolored nodes represent unregulated gene products which have a relationship with a colored node based on the literature. A line between two nodes represents a biological relationship with the line length indicating the amount of literature-based evidence supporting this relationship. The solid lines represent direct interactions while the dashed lines represent indirect interactions.</p