CARP knockdown in ARVMs induces sarcomere disarray.

<p><b>A:</b> ARVMs were transfected with 50 nM nonsilencing siRNA (top panel) or 50 nM CARP-siRNA (bottom panel and fixed at 48 h for immunofluorescence imaging. Cells were stained for CARP (green) myomesin (blue), and filamentous actin (red).</p

Doxorubicin inhibits CARP expression at the transcriptional level.

<p><b>A:</b> ARVMs were pretreated with 10 µg/ml cycloheximide (Cyclo), a protein synthesis inhibitor, in the presence or absence of 1 µM doxorubicin (Doxo) and cell lysates analyzed by immunoblot for CARP and actin and corresponding densitometry analysis is shown below. <b>B:</b> Comparison of CARP mRNA decay (quantified by RT-PCR) in ARVMs pretreated with 5 µg/ml actinomycin D (act D) in the presence or absence of 1 µM doxorubicin. <b>C:</b> NRVMs were transfected with a CARP promoter luciferase reporter (CARP-pGL3) and treated with increasing concentrations of doxorubicin. Cell lysates were assayed for luciferase activity and values were normalized to a promoterless control (pGL3 basic). Shown are mean±SD from 4 independent experiments. The qRT-PCR and luciferase-reporter experiments were performed in triplicate. * <i>P</i><0.05, ANOVA.</p

Sarcomeric and nuclear localization of CARP in ARVMs.

<p><b>A:</b> Low magnification (40×) images of ARVMs co-stained with antibodies to CARP (green) and myomesin (red). <b>B:</b> High magnification (100×) of ARVMs immunostained for CARP (green) and myomesin (red, left image) or α-actinin (red, right image), and shown below the image is the corresponding fluorescence intensity along the white arrow in the red and green channels.</p

GATA4 is an upstream regulator of CARP.

<p><b>A:</b> HEK-293 cells were cotransfected with CARP-pGL3 promoter (filled bars) and increasing concentrations of GATA4 expression plasmids and cell lysates were assayed for luciferase activity. The experiment was repeated with a promoterless pGL3 vector (open bars) as control. <b>B:</b> NRVMs were transfected with CARP-pGL3 along with either GATA4-siRNA, CARP-siRNA, or AdV-CARP for 24 h and cell lysates were assayed for luciferase activity. All luciferase-reporter experiments were performed in triplicate. Values were normalized to untreated pGL3 basic and shown as mean±SD from 4 independent experiments. <b>C:</b> Representative CARP immunoblot of NRVMs transfected with nonsilencing control or GATA4-siRNA for 24 or 48 h. <b>D:</b> Representative immunofluorescent images of NRVMs transfected with nonsilencing control or GATA4-siRNA for 48 h. NRVMs were co-stained for myomesin (green), filamentous actin (red), and DAPI (blue).</p

Doxorubicin decreases CARP levels in ARVMs.

<p><b>A:</b> ARVMs were treated for 24 h with increasing concentrations of doxorubicin and cell lysates analyzed by immunoblot for CARP and actin. Also shown is a time course of CARP and tubulin expression in ARVMs treated with 1 µM doxorubicin. CARP densitometry values for the time-course were normalized to tubulin and expressed as a percentage of the 0 time-point. Shown are mean±SD from 4 independent experiments. * <i>P</i><0.05 relative to 0 time-point. <b>B:</b> Cells were harvested and fractionated at various time points into nuclear and cytoplasmic extracts and analyzed for CARP expression by immunoblot. CARP densitometry values were normalized to tubulin (cytoplasmic fraction) or topoisomerase (nuclear fraction) and expressed as a percentage of the 0 time-point.</p

Calpain inhibition preserves titin but not CARP levels.

<p>ARVMs were treated for 24 h with 1 µM doxorubicin and cell lysates analyzed for <b>A:</b> titin in Coomassie stained agarose gels (representative of 4 independent experiments, arrowheads indicate titin degradation product), and <b>B:</b> CARP and tubulin by immunoblot. <b>C:</b> Bar graph shows corresponding quantification of CARP immunoblot analysis (% of control, n = 3) and myofibrillar disarray (% of cells scored for >50% disruption of myomesin striations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035743#s2" target="_blank">Methods</a>, n = 4–5, ∼150 cells counted per experiment). Shown are mean±SD, <i>P</i><0.05 relative to control (<b>*</b>) and Doxo (<b>†</b>).</p

CARP siRNA knockdown in ARVMs.

<p><b>A:</b> ARVMs were transfected for 24 h with increasing concentrations of CARP targeted siRNA and lysates analyzed by immunoblot for CARP and tubulin. Also shown is a time course of CARP and tubulin levels in ARVMs transfected with 50 nM CARP- siRNA. <b>B:</b> Cells were harvested at various time-points and fractionated into nuclear and cytoplasmic extracts and analyzed for CARP expression by immunoblot. CARP densitometry values were normalized to tubulin or topoisomerase and expressed as a percentage of the 0 time-point.</p

GATA4 overexpression in NRVM results in partial rescue of doxorubicin-induced sarcomere disarray.

<p><b>A:</b> Representative immunoblots for CARP and GATA4 from NRVMs infected with AdV-GATA4 and treated with doxorubicin in the presence or absence of CARP-siRNA. <b>B:</b> Corresponding densitometry values normalized to control are shown for CARP (open bars) and GATA4 (filled bars). Shown are mean±SD, n = 6, <i>P</i><0.05 relative to control (<b>*</b>), Doxo only (<b>§</b>), and Doxo+AdV-GATA4 (<b>†</b>) <b>C:</b> Immunofluorescent images of NRVMs treated with 0.5 µM doxorubicin alone or infected with AdV-GATA4 for 24 h followed by doxorubicin for 24 h. NRVMs were stained for myomesin (green), filamentous actin (red), and DAPI (blue). <b>D:</b> Bar graph shows % sarcomere disarray (n = 5–8, ∼150 cells counted per experiment). Values shown as mean±SD, <i>P</i><0.05 relative to control (<b>*</b>) and Doxo only treatment (<b>§</b>).</p

CARP and GATA4 regulate titin and actin transcription.

<p>NRVMs were cotransfected with either a titin promoter reporter (<b>A</b>) or actin promoter (<b>B</b>) along with 0.5 µM doxorubicin, CARP-siRNA, or GATA4-siRNA. Luciferase-reporter experiments were performed in triplicate. Values were normalized to untreated pGL3 basic and shown as mean±SD from 4 independent experiments.</p

Preservation of CARP in NRVM does not rescue doxorubicin-induced sarcomere disarray.

<p><b>A:</b> NRVMs were treated for 24 h with increasing concentrations of doxorubicin and cell lysates analyzed by immunoblot for CARP, GATA4, and tubulin. <b>B:</b> NRVMs were treated with 0.5 µM doxorubicin alone or infected with adenoviral CARP (AdV-CARP) for 24 h followed by doxorubicin treatment for 24 h and cell lysates were analyzed by immunoblot for CARP and tubulin. <b>C:</b> Immunofluorescent images of untreated control, NRVMs treated with 0.5 µM doxorubicin alone or NRVM infected with AdV-CARP for 24 h followed by doxorubicin for 24 h. NRVMs were co-stained for myomesin (green) and filamentous actin (red). Note: the purple appearance of nuclei is due to the overlay of DAPI staining with red autofluorescence from doxorubicin intercalated into nuclear DNA.</p