Journal: Molecular Carcinogenesis

Article Title: Molecular Insights Into Canine Hepatocellular Carcinoma: A Cross‐Species Transcriptomic Comparison With Human HCC

doi: 10.1002/mc.70092

Figure Lengend Snippet: The expression levels of ABCB4 and APOA1 in normal liver tissues and canine HCC clinical tissues. (A) Box and whiskers plot representing the relative expression of ABCB4 in canine HCC tissues (normal liver tissues, n = 6; HCC, n = 16) and (B) Box and whiskers plot representing the relative expression of APOA1 in canine HCC tissues (normal liver tissues, n = 6; HCC, n = 16). Statistical differences between normal liver and HCC tissues were evaluated using the Mann–Whitney U test. The y‐axis represents the relative expression level of respective mRNAs normalized with GAPDH in log10 units. ** p < 0.01, *** p < 0.001.

Article Snippet: Information on the TaqMan primer sequences for apolipoprotein A1 (APOA1, assay ID: Cf02682463), ATP‐binding cassette subfamily B member 4 (ABCB4, assay ID: Cf02647380), Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, assay ID: Cf04419463) is available at: https://www.thermofisher.com/order/genome-database/ .

Techniques: Expressing, MANN-WHITNEY

Journal: Molecular Carcinogenesis

Article Title: Molecular Insights Into Canine Hepatocellular Carcinoma: A Cross‐Species Transcriptomic Comparison With Human HCC

doi: 10.1002/mc.70092

Figure Lengend Snippet: Protein–protein interaction (PPI) network, hub gene identification, and module analysis from the DEGs in canine HCC. (A) PPI networks of the 174 differentially expressed mRNAs, consisting of 196 edges and 146 nodes. Here, pink node indicates the validated downregulated gene (APOA1) in the present study, while the upregulated gene (ABCB4) did not show interaction with other genes. The PPI network was constructed via Cytoscape v3.10.3. The size of the node indicates the degree of connectivity of a specific gene in the PPI network. (B) The PPI network of the top 10 hub genes, visualized by Cytoscape. The up and downregulated genes are shown by a color gradient from green to pink, respectively. (C) Detailed information on the top 10 hub genes in the PPI network. (D–F) Top 3 modules of the PPI network: module 1 with 30 nodes (D), module 2 with 14 nodes (E), and module 3 with 31 nodes (F). (G–I) KEGG pathways, exhibited by module 1 (G), module 2 (H), and module 3 (I). KEGG pathways were formulated using DAVID bioinformatics.

Article Snippet: Information on the TaqMan primer sequences for apolipoprotein A1 (APOA1, assay ID: Cf02682463), ATP‐binding cassette subfamily B member 4 (ABCB4, assay ID: Cf02647380), Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, assay ID: Cf04419463) is available at: https://www.thermofisher.com/order/genome-database/ .

Techniques: Construct

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: ApoA1 production following AAV2-apoA1(WT) and AAV2-apoA1(4WF) transduction assayed by ELISA and immunofluorescence. ( A ) Human embryonic kidney cells (HEK-293), rat smooth muscle cells (SMC), rat blood outgrowth endothelial cells (BOEC), and murine macrophages (Raw 264.7) were transduced with AAV2-apoA1(WT) and AAV2-apoA1(4WF). ApoA1 production was determined by ELISA of the media conditioned by transduced cells 3 days post-transduction. ( B ) Rat endothelial cells (a-c) and rat smooth muscle cells (d-f) were transduced with either AAV2-apoA1(WT) (a and d) or AAV2-apoA1(4WF) (b, e and f) for 3 days, methanol-fixed and reacted with anti-apoA1 antibody except (c), followed by either Cy 3.5- (a, b and c) or AlexaFluor488-labeled (d, e and f) secondary antibodies. Original magnification is 100×.

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: ApoA1(4WF)-, but not apoA1(WT)-mediated efflux following transduction with the respective AAV2 vectors demonstrates oxidative resistance. Cell culture media conditioned by HEK 293 cells transduced with AAV2-apoA1(WT), AAV2-apoA1(4WF), both containing 10 µg/ml of AAV2-encoded apoA1 variants, and the conditioned media from the non-transduced HEK-293 cells supplemented with 10 µg/ml of recombinant human apoA1 (rhapoA1) were incubated with increasing concentrations of HOCl. Cholesterol efflux assays were then performed on 3 H-cholesterol-loaded Raw 264.7 cells incubated with differently oxidized conditioned media from HEK-293 cells for 4 h. *P < 0.05 4WF vs WT. ✞P < 0.05 4WF vs rhapoA1.

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Cell Culture, Recombinant, Incubation

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: Impact of AAV2-apoA1(WT) and AAV2-apoA1(4WF) transduction on the proliferation and migration of SMC and BOEC, reactive oxygen species (ROS) production, and anti-inflammatory responses in endothelial cells. WST-8 assay was used to evaluate the effect of apoA1(WT) and apoA1(4WF) overexpression on proliferation of ( A ) TNFα (20 ng/ml)-stimulated rat aortic SMC and ( B ) non-stimulated rat BOEC. The results (both A and B ) were normalized by WST-8 assay values produced in confluent cultures of rat SMC ( A ) and rat BOEC ( B ) and expressed as the percent of a monolayer confluency. A monolayer scratch injury assay was used to assess migratory capacity of apoA1(WT)- and apoA1(4WF)-transduced rat SMC ( C ) and rat BOEC ( D ) compared with non-transduced counterparts. A closure of the gap inflicted by pipette tip was quantified 24 h after the scratch injury. ( E ) CM-H2DCFDA assay in non-transduced, apoA1(WT)- and apoA1(4WF)-transduced RAEC with/without TNFα stimulation. ( F ) Attachment of PKH26-labeled rat monocytes to TNFα-stimulated RAEC.

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Migration, Over Expression, Produced, Transferring, Labeling

Journal: Scientific Reports

Article Title: Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

doi: 10.1038/s41598-022-09524-y

Figure Lengend Snippet: Long-term expression of the therapeutic transgene, the anti-AAV2 neutralizing antibodies dynamics, and the morphometric results in hypercholesterolemic diabetic pigs. ( A ) The percent of retained AAV2eGFP transduction capacity after incubation with 1:20 diluted serum from experimental pigs at different time points throughout the study. NAB positivity was denoted at 50% reduction of transducibility of sera-incubated compared to the not incubated AAV2 vectors. ( B ) Representative images of Verhoeff-van Gieson – stained sections of a normal pig coronary artery and pig coronary arteries treated with BMS, AAV2-eGFP- and AAV2-apoA1(4WF)-eluting stents. ( C ) Morphometric measurements of the stented segments using 3 different calculation methods (percent of luminal stenosis, neointimal thickness, and the ratio of the neointimal area to the medial area).

Article Snippet: Human apoA1 ELISA assay was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Transduction, Incubation, Staining

Journal: Journal of Biological Chemistry

Article Title: CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells

doi: 10.1074/jbc.m116.714279

Figure Lengend Snippet: FIGURE2.CCL2inhibited[3H]cholesteroleffluxtoHDL/apoA1inECs.A,basalcholesteroleffluxtoHDL/apoA1inECs.HCAECsandHUVECswereloadedwith [3H]cholesterol (1 Ci/ml) for 24 h. Efflux was initiated by BSA alone and BSA plus 20 g/ml apoA1 or 50 g/ml HDL for 2 h. The radioactivity of the medium and cells was measured with a liquid scintillation counter. The cholesterol efflux was expressed as the percentage of counts in the medium relative to the total counts for the medium and cells together. B, dose response of CCL2 on cholesterol efflux to HDL. Cells were initiated by 50 g/ml human HDL 2 h after treating with increasing doses (0, 20, 40, and 80 ng/ml) of CCL2. The final cholesterol efflux was calculated as the percentage of total [3H]cholesterol released into the mediumaftersubtractionofthevaluesobtainedintheabsenceofHDL.OtherproceduresarethesameasA.C,timecourseofCCL2oncholesteroleffluxtoHDL. Cells were incubated with 50 g/ml human HDL 2 h after treating with 40 ng/ml CCL2 for indicate times (0, 12, 18, and 24 h). Others were the same as A. D, dose response of CCL2 on cholesterol efflux to apoA1. Cells were initiated by 20 g/ml human apoA1 2 h after CCL2 treated with increasing doses. Other procedures werethesameasA.E,timecourseofCCL2oncholesteroleffluxtoapoA1.Cellswereincubatedby20g/mlhumanapoA12hafterCCL2treatmentfordifferent times. Other procedures were the same as A. The results were expressed as mean S.D. (n 3). The points represent the averages of three values. #, p 0.001 compared with the BSA alone in HCAEC; ##, p 0.001 compared with the BSA alone in HUVEC. *, p 0.05; **, p 0.01; ***, p 0.001 compared with the untreated cells.

Article Snippet: After washing and fixation, the cells were incubated with DAPI for 5 min. Then, the cells were washed three times for 10 min and examined using a confocal microscope Cholesterol Efflux to HDL /ApoA1 Assay—After 6 h of serum starvation, the cells were washed with PBS and labeled by incubation in ECM supplemented with 0.5% BSA containing [3H]cholesterol (1 Ci/ml; PerkinElmer Life Sciences) for 24 h. Cellular cholesterol efflux was initiated by the addition of ECM containing 0.2% BSA with 20 g/ml human apoA1 or 50 g/ml HDL in the presence or absence of CCL2 (40 ng/ml).

Techniques: Radioactivity, Incubation

Journal: Journal of Biological Chemistry

Article Title: CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells

doi: 10.1074/jbc.m116.714279

Figure Lengend Snippet: FIGURE 4. siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, and internalization and on cholesterol efflux to HDL/apoA1 in HCAECs. After using si-CCR2, si-CCR3, and si-CCR5 interference, HCAECs were induced with 40 ng/ml CCL2 for 18 h. 125I-HDL binding at 4 °C, 125I-HDL association at 37 °C, HDL internalization, and [3H]cholesterol efflux were determined as the method in Figs. 1 and 2. A, roles of chemokine receptors on 125I-HDL binding after CCL2 was induced. B, roles of chemokine receptors on 125I-HDL association after CCL2 was induced. NS, non-significance. C, roles of chemokine receptorsontheinternalizationofHDLphospholipidandproteinafterCCL2wasinduced(20).D,rolesofchemokinereceptorsontheperinuclearlocalization of HDL protein after CCL2 was induced. The details of the method were same as used in Fig. 1D (20). E, roles of chemokine receptors on [3H]cholesterol efflux to HDL after CCL2 was induced. F, roles of chemokine receptors on [3H]cholesterol efflux to apoA1. Non-transfected represents non-transfected cells, and NC represents negative control (NC)-transfected cells. The values were shown as the means S.D. of triplicate. *, p 0.05; **, p 0.01; ***, p 0.001 compared with the indicated group.

Article Snippet: After washing and fixation, the cells were incubated with DAPI for 5 min. Then, the cells were washed three times for 10 min and examined using a confocal microscope Cholesterol Efflux to HDL /ApoA1 Assay—After 6 h of serum starvation, the cells were washed with PBS and labeled by incubation in ECM supplemented with 0.5% BSA containing [3H]cholesterol (1 Ci/ml; PerkinElmer Life Sciences) for 24 h. Cellular cholesterol efflux was initiated by the addition of ECM containing 0.2% BSA with 20 g/ml human apoA1 or 50 g/ml HDL in the presence or absence of CCL2 (40 ng/ml).

Techniques: Binding Assay, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells

doi: 10.1074/jbc.m116.714279

Figure Lengend Snippet: FIGURE 6. p42/44 MAPK inhibition reversed CCL2 suppression on HDL binding, association, and internalization and [3H]cholesterol efflux to HDL/apoA1 in HCAECs. HCAECs were preincubated with 10 M U0126 for 30 min and then treated with 40 ng/ml CCL2 for 18 h. 125I-HDL binding at 4 °C, 125I-HDL association at 37 °C, HDL internalization, and [3H]cholesterol efflux were determined as the method in Figs. 1 and 2. A, effect of U0126 on the decrease of 125I-HDL binding at 4 °C induced by CCL2. B, effect of U0126 on the decrease of 125I-HDL association at 37 °C. C, effect of U0126 on the suppression of the internalization of HDL phospholipid and protein (20). D, effect of U0126 on the suppression of perinuclear distribution of HDL protein (63). E, effect of U0126 on the suppression of [3H]cholesterol efflux to HDL. F, effect of U0126 on the decrease of [3H]cholesterol efflux to apoA1. The results are represented as means S.D. of at least three individual experiments. *, p 0.05; ***, p 0.001 compared with the indicated group.

Article Snippet: After washing and fixation, the cells were incubated with DAPI for 5 min. Then, the cells were washed three times for 10 min and examined using a confocal microscope Cholesterol Efflux to HDL /ApoA1 Assay—After 6 h of serum starvation, the cells were washed with PBS and labeled by incubation in ECM supplemented with 0.5% BSA containing [3H]cholesterol (1 Ci/ml; PerkinElmer Life Sciences) for 24 h. Cellular cholesterol efflux was initiated by the addition of ECM containing 0.2% BSA with 20 g/ml human apoA1 or 50 g/ml HDL in the presence or absence of CCL2 (40 ng/ml).

Techniques: Inhibition, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells

doi: 10.1074/jbc.m116.714279

Figure Lengend Snippet: FIGURE 7. Proposed model for the impact of CCL2 on HDL internalization andcholesteroleffluxandregulatorymechanismsinHCAECs.CCL2binds to CCR2, activates p42/44 MAPK pathway, and leads to the suppression of HDL internalization and cholesterol efflux to HDL/apoA1. ApoA1, apolipopro- tein AI.

Article Snippet: After washing and fixation, the cells were incubated with DAPI for 5 min. Then, the cells were washed three times for 10 min and examined using a confocal microscope Cholesterol Efflux to HDL /ApoA1 Assay—After 6 h of serum starvation, the cells were washed with PBS and labeled by incubation in ECM supplemented with 0.5% BSA containing [3H]cholesterol (1 Ci/ml; PerkinElmer Life Sciences) for 24 h. Cellular cholesterol efflux was initiated by the addition of ECM containing 0.2% BSA with 20 g/ml human apoA1 or 50 g/ml HDL in the presence or absence of CCL2 (40 ng/ml).

Techniques:

Journal: PloS one

Article Title: Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

doi: 10.1371/journal.pone.0019787

Figure Lengend Snippet: Figure 1. Expression of human apoA-I in D3 mouse ESCs. Undifferentiated ESCs were cultured in the absence of a feeder layer and transduced with lentiviral particles containing the full-length wild type apoA-I cDNA under the control of CMV promoter. This expression cassette was linked to the IRES-GFP reported cassette for the identification of transduced cells, while the lentiviral particles contain- ing the empty construct served as a control. To evaluate the success of gene transfer, undifferentiated empty construct- and apoA-I-transduced cells were examined for expression of GFP (A). The secretion of apoA-I in the concentrated spent medium was determined by Western blot analysis using an antibody specific to apoA-I. doi:10.1371/journal.pone.0019787.g001

Article Snippet: Lentivirus-mediated gene transfer To exogenously express apoA-I in murine ESCs and human iPSCs, full-length cDNA of human apoA-I obtained from Origene (SC 110828, Origene technologies, Rockville, MD) was subcloned into the lentiviral vector pHRST-CMV-IRESGFP (pLV-GFP) with blunt-end-ligation to generate pHRSTCMV-apoA-I-IRES-GFP (pLV-apoA-I).

Techniques: Expressing, Cell Culture, Transduction, Control, Construct, Western Blot

Journal: PloS one

Article Title: Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

doi: 10.1371/journal.pone.0019787

Figure Lengend Snippet: Figure 2. Effect of apoA-I gene transfer on generation of beating embryoid body and cardiac cells. (A) Empty construct and apoA-I- transduced mouse ESCs were differentiated using the conventional ‘‘hanging-drop’’ method, and the resultant embryoid bodies (EBs) were plated onto gelatin coated plates. The occurrence of beating areas within the EBs was observed and counted for 8 days starting from the day of plating. (B) Percentage of ESC-derived cardiomyocytes (troponin-T positive cells) on day 8 as determined by flow cytometry. (C) Individual cardiomyocytes were isolated from the beating area of the EBs and identified with immunnohistochemistry using antibody specific to the cardiac troponin-T. (D) Cardiac maker gene expression in the embryoid bodies derived from empty construct- and apoA-I-transduced ESCs as revealed by real-time quantitative PCR analysis. MHCA: a-myosin heavy chain; MHCB: b-myosin heavy chain; MLC2V: myosin light chain 2 ventricular transcripts. Data shown as mean 6 SEM from at least 3 independent experiments, n = 3–5, *p,0.05; **p,0.005. doi:10.1371/journal.pone.0019787.g002

Article Snippet: Lentivirus-mediated gene transfer To exogenously express apoA-I in murine ESCs and human iPSCs, full-length cDNA of human apoA-I obtained from Origene (SC 110828, Origene technologies, Rockville, MD) was subcloned into the lentiviral vector pHRST-CMV-IRESGFP (pLV-GFP) with blunt-end-ligation to generate pHRSTCMV-apoA-I-IRES-GFP (pLV-apoA-I).

Techniques: Construct, Derivative Assay, Flow Cytometry, Isolation, Gene Expression, Real-time Polymerase Chain Reaction

Journal: PloS one

Article Title: Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

doi: 10.1371/journal.pone.0019787

Figure Lengend Snippet: Figure 3. apoA-I gene transfer activates the BMP4-SMAD1/5 signaling cascade in undifferentiated ESCs. (A) The mRNA levels of bone morphogenic protein 4 (BMP4), activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 2 (ALK2) in the undifferentiated ESCs transduced with empty construct or apoA-I were evaluated by real-time quantitative PCR analysis using ribosomal protein S16 as internal control. (B) The phosphorylation status and total protein levels of the SMAD proteins were evaluated with Western blot analysis using b-actin as loading control. (C) The densitometry quantification of the Western blot results. Data shown as mean 6 SEM from 3 independent experiments,* p,0.05; ** p,0.005. doi:10.1371/journal.pone.0019787.g003

Article Snippet: Lentivirus-mediated gene transfer To exogenously express apoA-I in murine ESCs and human iPSCs, full-length cDNA of human apoA-I obtained from Origene (SC 110828, Origene technologies, Rockville, MD) was subcloned into the lentiviral vector pHRST-CMV-IRESGFP (pLV-GFP) with blunt-end-ligation to generate pHRSTCMV-apoA-I-IRES-GFP (pLV-apoA-I).

Techniques: Transduction, Construct, Real-time Polymerase Chain Reaction, Control, Phospho-proteomics, Western Blot

Journal: PloS one

Article Title: Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

doi: 10.1371/journal.pone.0019787

Figure Lengend Snippet: Figure 4. Inhibition of the BMP4 signaling pathway abolished the pro-cardiogenic effects of apoA-I gene transfer. The BMP4 signaling pathway was inhibited by the application of noggin (1 m/ml) to the apoA-I- transduced ESCs upon plating. (A) The phosphorylation status of the differentiating cells was evaluated 24 hr after noggin treatment. (B) The appearance of beating clusters during the 8-day period of differentiation. (C) The percentage of troponin-T positive cells as determined by flow cytometry analysis. Data shown as mean 6 SEM from 3 independent experiments, # p,0.05 comparing to the LV-apoA-I group; *p,0.05 and ** p,0.005 comparing to the LV-GFP group. doi:10.1371/journal.pone.0019787.g004

Article Snippet: Lentivirus-mediated gene transfer To exogenously express apoA-I in murine ESCs and human iPSCs, full-length cDNA of human apoA-I obtained from Origene (SC 110828, Origene technologies, Rockville, MD) was subcloned into the lentiviral vector pHRST-CMV-IRESGFP (pLV-GFP) with blunt-end-ligation to generate pHRSTCMV-apoA-I-IRES-GFP (pLV-apoA-I).

Techniques: Inhibition, Phospho-proteomics, Flow Cytometry

Journal: PloS one

Article Title: Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

doi: 10.1371/journal.pone.0019787

Figure Lengend Snippet: Figure 5. Synergistic effect of recombinant apoA-I and BMP4 on the cardiac differentiation of D3 ESCs. Untransduced D3 ESCs were subjected to cardiac differentiation in the presences or absence of recombinant apoA-1 (100 nM) and/or BMP4 (0.5 ng/ml). The appear- ance of beating clusters during the 8-day period of differentiation were recorded. Data shown as mean 6 SEM from 3 independent experiments, ** p,0.005 comparing to the control group. doi:10.1371/journal.pone.0019787.g005

Article Snippet: Lentivirus-mediated gene transfer To exogenously express apoA-I in murine ESCs and human iPSCs, full-length cDNA of human apoA-I obtained from Origene (SC 110828, Origene technologies, Rockville, MD) was subcloned into the lentiviral vector pHRST-CMV-IRESGFP (pLV-GFP) with blunt-end-ligation to generate pHRSTCMV-apoA-I-IRES-GFP (pLV-apoA-I).

Techniques: Recombinant, Control

Journal: PloS one

Article Title: Exogenous expression of human apoA-I enhances cardiac differentiation of pluripotent stem cells.

doi: 10.1371/journal.pone.0019787

Figure Lengend Snippet: Figure 6. Calcium handling property of the cardiomyocytes derived from empty construct- and apoA-I-transduced ESCs. (A) Representative tracings of rhythmic spontaneous Ca2+ transients in cardiomyocytes derived from empty construct- and apoA-I- transduced cells. (B): Amplitude, (C) Maximal upstroke velocity (Vmax upstroke), (D) Maximal decay velocity (Vmax decay) of Ca2+ transients in the mESC-derived cardiomyocytes. (E) Representative tracings of caffeine-induced Ca2+ release from sarcoplasmic reticulum in cardiomyocytes derived from wild type, empty construct and apoA-I-1a transduced cells (right), demonstrating caffeine-sensitive Ca2+ stores and fractional release of total sarcoplasmic reticulum Ca2+ load during spontaneous activation. (F): Amplitude, (G) Maximal upstroke velocity (Vmax upstroke), (H) Maximal decay velocity (Vmax decay) of Ca2+ transients in the ESC-derived cardiomyocytes. Data shown as mean 6 SEM from the recordings of 20–30 cells from 3–5 independent experiments, * p,0.05; ** p,0.005. doi:10.1371/journal.pone.0019787.g006

Article Snippet: Lentivirus-mediated gene transfer To exogenously express apoA-I in murine ESCs and human iPSCs, full-length cDNA of human apoA-I obtained from Origene (SC 110828, Origene technologies, Rockville, MD) was subcloned into the lentiviral vector pHRST-CMV-IRESGFP (pLV-GFP) with blunt-end-ligation to generate pHRSTCMV-apoA-I-IRES-GFP (pLV-apoA-I).

Techniques: Derivative Assay, Construct, Activation Assay