human pasmcs (hpasmcs  (Thermo Fisher)

Journal: Physiological Reports

Article Title: The effect of mitoTEMPO on the development of hypoxia‐induced pulmonary hypertension in male mice

doi: 10.14814/phy2.70804

Figure Lengend Snippet: Effect of mitoTEMPO treatment on HIF‐1α stabilization in vitro. HIF‐1α protein levels were assessed by western blot, and steady‐state mRNA levels for lactate dehydrogenase A ( Ldha ) and pyruvate dehydrogenase kinase 1 ( Pdk1 ) by quantitative real‐time PCR in (a) CMT167 cells, (b) hPASMCs, and (c) mPASMCs, exposed to normoxia (21% O 2 ), severe hypoxia (1% O 2 ), or mild hypoxia (10% O 2 ) for 24 h and treated with triphenylphosphonium (TPP + ) (blue dots) or mitoTEMPO (MT) (red dots). Immunoblots shown are representative of three independent experiments. CMT167: mouse lung carcinoma epithelial cells; hPASMCs: human pulmonary artery smooth muscle cells; mPASMCs: mouse pulmonary artery smooth muscle cells. Densitometric analysis of HIF‐1α bands normalized to β‐Actin. Data are presented as mean ± SD. Statistical comparisons were made using two‐way ANOVA with Tukey's post hoc test ( n = 3 per group). (ns: no signal). Quantitative real‐time PCR data reflect mean ΔCt ± SD ( n = 3 per experimental group).

Article Snippet: Mouse lung carcinoma epithelial (CMT167) cells (10032302, Merck, Germany) and human PASMCs (hPASMCs) (C‐12521, PromoCell, Germany) were purchased.

Techniques: In Vitro, Western Blot, Real-time Polymerase Chain Reaction

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunocytochemistry images of pSPHK2 (pink), actin (green, cytoplasmic marker) and DAPI (blue, nuclear) coimmunostaining in EMAP II treated (2 hr) or vehicle treated fixed hPASMCs, scale bar is 20 μm, n=3. (B) Representative immunoblot probed for pSPHK2, tubulin and lamin B in cytoplasmic and nuclear fractions of hPASMCs following EMAP II treatment for 0, 2 and 4 hours, n=3. (C) Representative immunoblot probed for pSPHK2 and lamin B in nuclear fractions of hPASMCs following EMAP II treatment (150 minutes) with or without SPHK2 inhibitor (D) quantification of nuclear pSPHK2/lamin B, n=3. (E) ELISA-nuclear C18-S1P levels normalized against 1 μg of nuclear proteins in the nuclear fractions of hPASMCs following EMAP II for 15 or 150 minutes with or without SPHK2 inhibitor, n=3 or 4/group. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Immunocytochemistry, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Schematic diagram representing the collection of vascular endothelial cells (ECs) conditioned media (ECM) from ECs grown in 1%O2 or room air to treat vascular smooth muscle cells (SMCs) and, (B) representative dot blot probed for secreted EMAP II expression in ECM. (C) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing and, (D) quantification of KLF4/Tubulin, n=3 and (E) quantification of Ac-H3K9/total histone H3, n=3. (F) KLF4 expression levels normalized against 18S rRNA in normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing, n=3–4. (G) EMAP II secreted by vascular ECs promote SPHK2/Ac-H3K9/KLF4 signaling in vascular SMCs that may promote PASMCs proliferation. P values are calculated using Kruskal-Wallis against Hy ECM+Scr or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Dot Blot, Expressing, Western Blot, Transfection

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for AIMP1 (precursor form of EMAP II) and Tubulin in protein lysates of human iPAH or FDL, n=19–20/group and, (B) quantitation of AIMP1 (AIMP1/Tubulin) in protein lysates of human iPAH or FDL, n=19–20/group. (C) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPASMCs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (D) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPASMCs, n=4. (E) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPMVECs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (F) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPMVECs, n=3. P values are calculated using unpaired t-test or Kolmogorov-Smirnov non-parametric testing and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for Ac-H3K9, total H3, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 4 hours and (B) quantitation of Ac-H3K9/total H3 and (C) quantification of SPHK2/tubulin, n=4. (D) Volcano plot showed the log2-fold changes and statistical significance of hyperacetylated H3K9 regions calculated after differential binding analysis of EMAP II treated vs control hPASMCs. Pink points indicate significantly hyperacetylated H3K9 regions in EMAP II (right to 0) or in control (left to 0). FDR=0.05, n=2 (E) Genome wide distribution of differentially enriched hyperacetylated H3K9 peaks (log2-fold change > 1, p value < 0.05) n=2. (F) Number of peaks of Ac-H3K9 normalized to IgG in with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs, n=2. (G) Gene Ontology results using differentially enriched Ac-H3K9 peaks in EMAP II treated hPASMCs, n=2. (H) Cell proliferation rate in hPASMCs treated with vehicle or EMAP II following SPHK2 inhibitor treatment for 24 hours, n=3. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Transfection, Quantitation Assay, Binding Assay, Control, Genome Wide

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) The Venn’s diagram of differential acetylated sites in control vs EMAP II (total) (purple), EMAP II vs iSPHK2+EMAP II (yellow) and control vs EMAPII only in 5’UTR and upstream with fold enrichment greater than 2 (green). The red circle indicates the potential gene set with potential upstream candidate regulatory elements that would be differentially acetylated by EMAP II through SPHK2 in hPASMCs. Venn diagram is created using Venny 2.1 (an online interactive tool), n=2/group (B) Snapshot of IGV view of KLF4 gene in Ac-H3K9 CUT&RUN data of with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs. (cCRE= candidate Cis-Regulatory Elements) n=2/group (C) Representative immunoblot probed for KLF4, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 6–8 hours, and (D) quantitation of KLF4/tubulin and (E) KLF4 expression levels normalized against 18S rRNA in hPASMC cells following siRNA mediated SPHK2 silencing and EMAP II treatment for 6 hours, n=4. P values are calculated using Kolmogorov-Smirnov non-parametric testing and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Control, Western Blot, Transfection, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) RNA-seq data of SPHK2, KLF4 and AIMP1 in iPAH: PASMCs and non-iPAH:PASMCs in log2-fold of count per million (cpm). Following two-way ANOVA, Sidak’s multiple comparisons test for logarithmic values, n=4. (B) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection and, quantification of (C) Ac-H3K9/total histone H3 and, (D) KLF4/Tubulin, n=3 (E) KLF4 expression levels normalized against 18S rRNA in non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection, n=4. (F) Cell proliferation rate of non: iPAH or iPAH PASMC with or without iSPHK2 pretreatment for 24 hours, n=4. (G) The proposed model: Endothelial monocyte activating polypeptide II (EMAP II) plays a key role in reawakening pluripotency factor, KLF4 in human pulmonary artery smooth muscle cells (PASMCs) through stimulation of the nuclear SPHK2/S1P epigenetic modulating axis, suggesting that cooperation between SPHK2 and EMAP II could be a major driving force for epigenetic-mediated vascular PASMCs reprogramming and remodeling in PH. Ablation of SPHK2 expression confers protection against PH by rescuing the global and local transcription machinery from histone acetylation and activation of the pluripotency factor, KLF4. P values are calculated using Kruskal-Wallis against iPAH or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: RNA Sequencing, Western Blot, Transfection, Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1371/journal.pone.0037712

Figure Lengend Snippet: HPASMCs were serum starved (1% FBS treated) or 10% serum treated for 24 h, and then (A) EZH2 expression was detected by qRT–PCR. ( B ) EZH2 protein expression was analyzed in serum-starved or 10% serum-treated HPASMCs by Western blotting. All experiments were repeated at least 3 independent times. * P <0.05 compared with 5% FBS.

Article Snippet: Human PASMCS (HPASMCs) were purchased (Cascade Biologics, Portland, OR) and maintained in growth media M231(Invitrogen, Carlsbad, CA) supplemented with Smooth Muscle Growth Supplement (SMGS) (Invitrogen, Carlsbad, CA) at 37°C in 5% CO 2 as described in earlier studies .

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1371/journal.pone.0037712

Figure Lengend Snippet: HPASMCs were transfected with a plasmid expressing EZH2 or control plasmids using 4D-Nucleofector system. A) EZH2 protein expression was analyzed by western blot (upper panel) and mRNA expression was analyzed by real-time PCR (lower panel) to determine the transfection efficiency. B) The transfected cells were fixed and labeled with PI after 48 hrs and analyzed for cell cycle by flow cytometry. Percentage number of cells in S and G2/M phase of the cell cycle is plotted (upper panel) and representative histograms from the cell cycle analysis of EZH2 and control GFP plasmid transfected cells are shown (lower panel). C) Real-time PCR analysis of cell proliferation markers expressed as normalized fold expression is shown. All experiments were repeated at least 3 times and graphs shown are average of three independent experiments.

Article Snippet: Human PASMCS (HPASMCs) were purchased (Cascade Biologics, Portland, OR) and maintained in growth media M231(Invitrogen, Carlsbad, CA) supplemented with Smooth Muscle Growth Supplement (SMGS) (Invitrogen, Carlsbad, CA) at 37°C in 5% CO 2 as described in earlier studies .

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cell Cycle Assay

Journal: PLoS ONE

Article Title: Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1371/journal.pone.0037712

Figure Lengend Snippet: The HPASMCs were transfected with EZH2 and GFP plasmid or only with GFP plasmid (Control). An artificial wound was created using a 1 ml pipette on a confluent monolayer of cells. A) Images were taken at 0 and 24 hrs after wound. B) Migration was quantified visually as the number of cells migrating in to the gap and average of three experiments is plotted.

Article Snippet: Human PASMCS (HPASMCs) were purchased (Cascade Biologics, Portland, OR) and maintained in growth media M231(Invitrogen, Carlsbad, CA) supplemented with Smooth Muscle Growth Supplement (SMGS) (Invitrogen, Carlsbad, CA) at 37°C in 5% CO 2 as described in earlier studies .

Techniques: Transfection, Plasmid Preparation, Transferring, Migration

Journal: PLoS ONE

Article Title: Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1371/journal.pone.0037712

Figure Lengend Snippet: HPASMCs cells were transfected with a plasmid expressing EZH2 or with GFP using 4D-Nucleofector system. Apoptosis was measured in transfected cells by using Annexin V staining. Propidium iodide (PI) incorporation was measured by flow cytometry to assess the mode of cell death. Relative fluorescence units represent the intensity of annexin V incorporation and PI incorporation was quantified. The noted experiments are representative of a minimum of four similar evaluations. * P <0.05 compared with control.

Article Snippet: Human PASMCS (HPASMCs) were purchased (Cascade Biologics, Portland, OR) and maintained in growth media M231(Invitrogen, Carlsbad, CA) supplemented with Smooth Muscle Growth Supplement (SMGS) (Invitrogen, Carlsbad, CA) at 37°C in 5% CO 2 as described in earlier studies .

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

doi: 10.1371/journal.pone.0037712

Figure Lengend Snippet: The HPASMCs were transfected with EZH2 or control plasmid and expression levels of calponin, a SMC phenotypic marker was assessed for A) mRNA by real-time PCR and B) protein levels by Western blot. All experiments were repeated at least 3 times and graph plotted is an average of three independent experiments. P<0.005 compared to controls. C) EZh2 increases binding to calponin-1 promoter in HPASMCs. DNA fragments interacting with H3K27me3 were pulled down by anti H3K27me3 or normal rabbit antibody. The presence of Calponin promoter DNA was checked by PCR using calponin promoter specific primers (upper panel). MYOD1 promoter segment binding to H3K27me3 was used as a positive control (lower panel). Calponin promoter region was amplified in EZH2 transfected cells and immunoprecipitated with anti-H3K27me3 but not in vector transfected cells (lanes 2 and 5). The immnoprecipitation with normal rabbit IgG did not yield any PCR amplification (lanes 3 and 6).

Article Snippet: Human PASMCS (HPASMCs) were purchased (Cascade Biologics, Portland, OR) and maintained in growth media M231(Invitrogen, Carlsbad, CA) supplemented with Smooth Muscle Growth Supplement (SMGS) (Invitrogen, Carlsbad, CA) at 37°C in 5% CO 2 as described in earlier studies .

Techniques: Transfection, Plasmid Preparation, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Positive Control, Amplification, Immunoprecipitation