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: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: CFZ-induced cell killing is ubiquitin-dependent. HPASMCs were transfected with control siRNA and Ub siRNA. (A) Knocking down efficiency was monitored by western blotting. (B) After transfection for 48 h, the transfection medium was changed to the starvation medium. Four hours later, cells were treated with CFZ (0.3 μM) for 20 h. The cell number was determined by counting on a haemocytometer. (C and D) Cell lysates were subjected to immunoblotting to analyse the expression of p62 and Bcl-xL. (E) Cell lysates were immunoprecipitated (IP) with TP53INP1 antibody, followed by immunoblotting with the LC3B antibody. The bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05 (n = 4).

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Ubiquitin Proteomics, Transfection, Control, Western Blot, Expressing, Immunoprecipitation

Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: CFZ induces autophagic cell death. (A and B) Pulmonary arteries (PAs) were surgically isolated from rats subjected to SU5416/hypoxia treatment with remodelled PA and from normal rats, homogenized, and subjected to western blotting analysis for LC3B-II and p62 expression. Bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (C–E) HPASMCs were transfected with control siRNA and LC3B, AMPK or TP53INP1 siRNA. After 48 h, transfection medium were changed to starvation medium for 4 h, and then HPASMCs were treated with CFZ (0.3 μM) for 20 h. Bar graphs show the cell number determined by counting on a haemocytometer (LC3B siRNA, n = 6; AMPK siRNA, n = 4; TP53INP1, n = 4). *Values significantly different from each other at P< 0.05. (F) HPASMCs were treated with CFZ (0.3 μM) for 20 h, and cell lysates were immunoprecipitated (IP) with TP53INP1 antibody, followed by immunoblotting with the LC3B antibody. The bar graph represents means ± SEM. *Values significantly different from each other at P< 0.05(n = 3).

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Isolation, Western Blot, Expressing, Control, Transfection, Immunoprecipitation

Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: CFZ induces protein ubiquitination. (A) Isolated pulmonary arteries from the SU5416/hypoxia model of PAH were homogenized, and subjected to western blotting to monitor protein ubiquitination using a ubiquitin (Ub) antibody. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (B) Protein ubiquitination was monitored in isolated pulmonary arteries from the SU5416/ovalbumin model of PAH. (PAH, n = 6; PAH + CFZ, n = 6). Bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05. (C) HPASMCs were treated with various doses of CFZ for 20 h. Cell lysates were analysed for protein ubiquitination by western blotting. (D) Cells were treated with CFZ (0.3 μM) for various durations. Bar graphs represent means ± SEM. *Values significantly different from untreated control at P< 0.05 (n = 6).

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Ubiquitin Proteomics, Isolation, Western Blot, Control

Journal: Cardiovascular Research

Article Title: Carfilzomib reverses pulmonary arterial hypertension

doi: 10.1093/cvr/cvw047

Figure Lengend Snippet: Identifications of proteins that are ubiquitinated by CFZ. (A) HPASMCs were treated with CFZ (0.3 μM) for 20 h, and cell lysates were immunoprecipitated (IP) with the Ub antibody. Bands indicated by arrows in Coomassie Blue-stained gels were consistently increased by CFZ treatment. The bar graph represents means ± SEM (n = 3). Mass spectrometry identified that these bands contain MVP and HSP90. Ubiquitination of MVP was confirmed in (B) CFZ-treated HPASMCs (n = 6), (C) isolated pulmonary arteries from SU5416/ovalbumin model (n = 4), and (D) isolated pulmonary arteries from SU5416/hypoxia model (n = 4) by immunoprecipitation with Ub antibody followed by immunoblotting with MVP. The ubiquitination of HSP90 were confirmed in (E) CFZ-treated HPASMCs (n = 6), (F) isolated pulmonary arteries from SU5416/ovalbumin model (n = 6), and (G) isolated pulmonary arteries from SU5416/hypoxia model (n = 4) by immunoprecipitation with Ub antibody followed by immunoblotting with HSP90 antibody. (H and I) HPASMCs were transfected with siRNA to knockdown MVP or HSP90. Efficiency of siRNA knockdown is shown in the western blotting data. Cells were treated with CFZ (0.3 μM) for 20 h, and the cell number was determined by counting on a haemocytometer. (MVP siRNA, n = 5; HSP90 siRNA, n = 6). *Values significantly different from each other at P< 0.05.

Article Snippet: Human PASMCs (HPASMCs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C.

Techniques: Immunoprecipitation, Staining, Mass Spectrometry, Ubiquitin Proteomics, Isolation, Western Blot, Transfection, Knockdown