Simulated treatments of SMA fibroblasts with two different cAMP modulators.

<p>Dual dose simulation data for the combination treatments with forskolin and dbcAMP (<b>A</b>), rolipram and dbcAMP (<b>B</b>) and forskolin and rolipram (<b>C</b>).</p

Normalized local sensitivity of gem concentration to full cAMP:<i>SMN2</i> model parameters.

<p>Normalized local sensitivity of gem concentration to full cAMP:<i>SMN2</i> model parameters.</p

Effects of C5-substituted 2,4-DAQs on expression of <i>full-length SMN</i> (<i>FL-SMN</i>) and <i>SMNΔ7</i> mRNA levels in fibroblasts.

<p>Fibroblasts were treated with 1–1000 nM D156844, D158872, D157161, D157495 or DMSO for 5 days. mRNA levels of <i>FL-SMN</i> and <i>SMNΔ7</i> were measured via quantitative RT-PCR with <i>ACTB</i>, <i>GAPD</i> and <i>RPLP0</i> being used as reference transcripts. The levels of either <i>FL-SMN (A)</i> or <i>SMNΔ7 (B)</i> mRNAs were not affected by the compounds in GM03813 fibroblasts. All transcript levels were expressed relative to DMSO-treated, GM03813 cells (dashed line). The basal levels of <i>FL-SMN (C)</i> and <i>SMNΔ7 (D)</i> mRNAs were measured in 3 different type II SMA (GM03813, GM22592 and AIDHC-SP22) and non-SMA (AIDHC-NMC1, AIDHC-SC1 and AIDHC-SC2) fibroblast lines. All transcript levels were expressed relative to GM03813 cells (dashed line). These fibroblast lines were subsequently treated for 5 days with 1 μM D156844, D158872, D157161, D157495 or DMSO. An increase in either <i>FL-SMN (E)</i> or <i>SMNΔ7 (F)</i> mRNA levels was not observed in any cell line treated with these C5-substituted 2,4-DAQs. All transcript levels were expressed relative to DMSO-treated cells for each fibroblast line (dashed line).</p

Effects of C5-substituted 2,4-DAQs on <i>Smn</i> and <i>Atoh7</i> expression in NSC-34 cells.

<p>NSC-34 cells were treated with 1 μM D156844, D158872, D157161, D157495 or DMSO for 5 days (n = 3/compound). These compounds did not affect <i>Smn</i> (<i>A</i>) mRNA levels as measured by qRT-PCR. Treated NSC-34 cells were also measured for Smn protein levels by immunoblot. (<i>B</i>) Representative SMN and β-tubulin immunoblots of NSC-34 cells treated with 1 μM D156844, D158872, D157161, D157495 or DMSO for 5 days. (<i>C</i>) Relative Smn protein levels in NSC-34 cells treated with 2,4-DAQs. Smn protein levels were not increased by these compounds in NSC-34 cells. (<i>D</i>) <i>Atoh7</i> mRNA levels were measured in treated NSC-34 cells by qRT-PCR. All 4 compounds significantly increased <i>Atoh7</i> mRNA levels in NSC-34 cells. The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and DMSO-treated cells.</p

Effects of C5-substituted 2,4-DAQs on the mRNA expression of DcpS regulated transcripts.

<p><i>(A)</i> GM03813 fibroblasts were treated with 1 μM D156844, D158872, D157161, D157495 or DMSO for 5 days. mRNA levels of <i>ATOH7</i>, <i>DRNT1</i>, <i>DRNT2</i> and <i>PAQR8</i> were measured via quantitative RT-PCR with <i>ACTB</i>, <i>GAPD</i> and <i>RPLP0</i> being used as reference transcripts. All of the 2,4-DAQs tested increased <i>ATOH7</i>, <i>DRNT1</i> and <i>DRNT2</i> transcript levels in SMA fibroblasts. All transcript levels were expressed relative to DMSO-treated, GM03813 cells (dashed line). The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and DMSO-treated cells. The basal levels of <i>ATOH7 (B)</i> mRNA as well as <i>DRNT1 (C)</i> and <i>DRNT2 (D)</i> lncRNAs were measured in 3 different type II SMA (GM03813, GM22592 and AIDHC-SP22) and non-SMA (AIDHC-NMC1, AIDHC-SC1 and AIDHC-SC2) fibroblast lines. <i>ATOH7</i> mRNA levels were higher in non-SMA fibroblasts than in SMA fibroblasts. Under basal conditions, <i>DRNT1</i> and <i>DRNT2</i> lncRNA levels are not significantly different between type II SMA and non-SMA fibroblasts. All transcript levels were expressed relative to GM03813 cells (dashed line). The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference relative to GM03813 fibroblasts. These fibroblast lines were separately treated for 5 days with 1 μM D156844, D158872, D157161, D157495 or DMSO and monitored for changes in <i>ATOH7 (E)</i>, <i>DRNT1 (F)</i> and <i>DRNT2 (G)</i> transcript levels. Increases in <i>ATOH7</i>, <i>DRNT1</i> and <i>DRNT2</i> transcript levels were observed in all fibroblast lines treated with these C5-substituted 2,4-DAQs. All transcript levels were expressed relative to DMSO-treated cells for each fibroblast line (dashed line). The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and DMSO-treated cells.</p

Effects of C5-substituted 2,4-DAQs on <i>SMN2</i>-drived BLA activity.

<p>Clone 11 NSC-34 cells harboring a reporter gene driven by the 3.4-kb <i>SMN2</i> promoter were treated with 1 μM D156844, D158872, D157161, D157495 or DMSO (n = 4/drug) for 19 hours prior to fluorescent β-lactamase assay analysis. <i>(A)</i> All 4 compounds significantly increased <i>SMN2</i>-drived BLA activity. The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and vehicle-treated cells. Dose-response curves (1 nM– 10 μM) for D156844 <i>(B)</i>, D158872 <i>(C)</i>, D157161 <i>(D)</i> and D157495 <i>(E)</i>. Each compound tested exhibited a dose-dependent increase in <i>SMN2</i>-drived BLA activity.</p

Optimized parameter values for cAMP pathway alternate cAMP:<i>SMN2</i> model.

<p>The parameters marked with an asterisk (*) were carried over from the simplified cAMP model.</p><p>Optimized parameter values for cAMP pathway alternate cAMP:<i>SMN2</i> model.</p

Effects of C5-substituted 2,4-DAQs on SMN protein levels in fibroblasts.

<p><i>(A)</i> Representative SMN and β-actin immunoblots of GM03813 type II SMA fibroblasts treated for 5 days with 1–1000 nM D156844, D158872, D157161, D157495 or DMSO. <i>(B)</i> Relative SMN protein levels—expressed as the ratio between SMN and β-actin band intensities—in GM03813 fibroblasts treated with 2,4-DAQs. All SMN protein levels were expressed relative to DMSO-treated GM03813 fibroblasts. <i>(C)</i> Representative SMN and β-actin immunoblots of type II SMA and non-SMA fibroblasts treated for 5 days with 1 μM D156844, D158872, D157161, D157495 or DMSO. <i>(D)</i> Relative SMN protein levels in type II SMA and non-SMA fibroblasts treated with 1 μM 2,4-DAQs. All SMN protein levels were expressed relative to DMSO-treated cells for each fibroblast line (dashed line). The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and DMSO-treated cells.</p

The effects of cAMP signaling modulators on gem counts in SMA fibroblasts.

<p>GM03813 SMA fibroblasts were treated with differing doses of dbcAMP, epinephrine, forskolin, salbutamol or rolipram for 5 days (n = 3/dose/drug). The concentrations for each dose of each drug (in µM) are shown below the appropriate bars of each graph. The vehicle for dbcAMP is ddH₂O (black bars) while DMSO (grey) serves as vehicle for the remaining compounds. The number of SMN-positive nuclear gems was counted in 100 randomly selected nuclei. Gem count analysis was also completed in GM03814 carrier fibroblasts so as to compare the gem data in treated SMA fibroblasts to those observed in healthy cells. The gem count analysis was expressed as (<b>A</b>) the number of gems per 100 nuclei, (<b>B</b>) the proportion of cells containing gems and (<b>C</b>) the proportion of cells containing multiple gems. The asterisk (*) denotes a statistically significant (p≤0.05) difference between drug-treated cells and vehicle (either ddH₂O for dbcAMP or DMSO for epinephrine, forskolin, salbutamol or rolipram)-treated cells.</p

The effect of cAMP signaling modulators on SMN localization to gems.

<p>SMN immunostaining (red) of GM03813 SMA fibroblasts treated for 5 days with (<b>A</b>) 500 µM dbcAMP, (<b>B</b>) ddH₂O (vehicle for <b>A</b>), (<b>C</b>) 100 nM epinephrine, (<b>D</b>) 50 µM forskolin, (<b>E</b>) 100 nM salbutamol, (<b>F</b>) 10 µM rolipram or (<b>G</b>) DMSO (vehicle for <b>C-F</b>). (<b>H</b>) SMN immunostaining of GM03814 carrier fibroblasts. The nuclei were counterstained with Hoescht 33342 (blue). Scale bar, 13 µm.</p