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, tubulin and Ponceau S staining in protein lysates of human idiopathic pulmonary arterial hypertension (iPAH: type of Group 1 PH) lung or failed donor lung (FDL) tissue specimens and (B) quantitation of Ac-H3K9/Total H3, n=19–20 (C) quantitation of Ac-H3K9/Tubulin in protein lysates of human iPAH (n=11) or FDL (n=9). (D) SPHK2 expression levels normalized against 18S rRNA in iPAH lung and FDL tissues. n=20 (E) Representative immunoblot probed for SPHK2 and Tubulin in protein lysates of human iPAH (type of Group 1 PH) lung or FDL tissue specimens and (F) quantitation of SPHK2/Tubulin in protein lysates of human iPAH lung or FDL, n=20. P values are calculated using unpaired t-test and results are shown as means ± SEM.

Article Snippet: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

Techniques: Western Blot, Staining, 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: SPHK2 KO or wild type (WT) control mice (C57BL/6NJ) were subjected to 3 wks of hypoxia (10% O2) or normoxia (room air). (A) Pulmonary vascular resistance (the maximum velocity of tricuspid regurgitation/the velocity time integral of the right ventricular outflow tract, TRmax velocity/VTIRVOT) n=8–10/group. (B) Pulmonary acceleration time (PAT), n=8–10/group. (C) RV hypertrophy/Fulton Index (the weight ratio of the right ventricle divided by the sum of left ventricle and septum, RV/(LV + S)) n=8–10/group. (D) Representative images of elastin-stained distal pulmonary vessels and, wall thickness of distal pulmonary vessels in elastin-stained lung tissue sections (wall thickness (%) = (2 × medial wall thickness / external diameter) × 100) n= 3/group randomly selected mice per each group and n=15 images of distal pulmonary vessels, scale bar is 10 μm, n=5–6/group. (E) Representative immunoblot probed for Ac-H3K9, total H3, Tubulin and ponceau S staining in whole tissue lysates from WT or SPHK2 KO in normoxia or hypoxia on same blot n=5–7/group and, quantitation of Ac-H3K9/Total H3. P values are calculated using one-way ANOVA following Tukey’s multiple comparisons test, and results are shown as means ± SEM.

Article Snippet: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

Techniques: Control, Staining, Western Blot, Quantitation Assay

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: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

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: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

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 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: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

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: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

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: Immunoblotting confirmed the EMAPII induced nuclear expression of pSPHK2 while pretreatment with SPHK2 inhibitor [iSPHK2: ABC294640 (Echelon Biosciences Inc., Salt Lake City, UT)] of 10 μM for 1 hour prior to EMAPII treatment ablated the EMAPII induced nuclear activation of SPHK2 ( , ).

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

Journal: International journal of radiation oncology, biology, physics

Article Title: Decreasing the Toxicity of Radiation Therapy: Radioprotectors and Radiomitigators Being Developed by the National Cancer Institute Through Small Business Innovation Research Contracts

doi: 10.1016/j.ijrobp.2018.12.027

Figure Lengend Snippet: NCI’s SBIR Funded Contracts for Radioprotectors and Radiomitigators that Advanced to Phase II. Specific aims involving humans are bolded.

Article Snippet: Redhill Biopharma Ltd. (Tel Aviv, Israel) is currently pursuing the anti-cancer properties of ABC294640 (Yeliva ® ) and has initiated clinical trials in cholangiocarcinoma, hepatocellular carcinoma, and multiple myeloma ( https://clinicaltrials.gov/ ; {"type":"clinical-trial","attrs":{"text":"NCT03377179","term_id":"NCT03377179"}} NCT03377179 ; {"type":"clinical-trial","attrs":{"text":"NCT02939807","term_id":"NCT02939807"}} NCT02939807 ; {"type":"clinical-trial","attrs":{"text":"NCT02757326","term_id":"NCT02757326"}} NCT02757326 ).

Techniques: In Vitro, Irradiation, Clinical Proteomics, Inhibition, Ex Vivo, Cell Culture

Journal: American Journal of Cancer Research

Article Title: Targeting sphingosine kinase 2 suppresses cell growth and synergizes with BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation in cholangiocarcinoma

doi:

Figure Lengend Snippet: ABC294640 acts synergistically with BH3-mimetics in inhibiting cholangiocarcinoma cell growth. A. RBE, HCCC9810 and HuH28 cells were treated with various concentrations of ABC294640 or ABT-263 alone or their combination for 72 h, then the cell viability was analyzed by CCK-8 assay. The results are presented as mean ± SEM from 3-4 independent experiments. The combination index (CI) was determined using the Chou-Talalay Method. CI < 1 indicates that the interaction between ABC294640 and ABT-263 was synergistic. B. RBE, HCCC9810 and HuH28 cells were treated with various concentrations of ABC294640 or Obatoclax alone or their combination for 72 h, then the cell viability was analyzed by CCK-8 assay. The results are presented as mean ± SEM from 3-4 indepen-dent experiments. CI was determined using the Chou-Talalay Method. CI < 1 indicates that the interaction between ABC294640 and Obatoclax was synergistic.

Article Snippet: In this article, we discovered an unintentional error that the Combination Index (CI) image of ABC294640 with Obatoclax in HCCC9810 cells in was misplaced during the preparation of figures using GraphPad Prism 6.0 software.

Techniques: CCK-8 Assay

Journal: MedComm

Article Title: Reticulon 3 regulates sphingosine‐1‐phosphate synthesis in endothelial cells to control blood pressure

doi: 10.1002/mco2.480

Figure Lengend Snippet: Lack of RTN3 activates the S1P signaling pathway. (A) plasma sphingosine‐1‐phosphate (S1P) levels in WT mice and RTN3‐null mice. (B–D) Extracellular S1P (B), extracellular NO (C), and intracellular cGMP (D) in HUVECs transfected with RTN3 siRNA and controls. (E) The expression of RTN3 and SPHK2 in HUVECs with/without RTN3 siRNA transfection. (F–H) The levels of extracellular S1P (F), extracellular NO (G), and intracellular cGMP (H) in HUVECs overexpressing RTN3 and controls. (I) The expression of RTN3 and SPHK2 in HUVECs with over‐expressed RTN3 and controls. (J–L) The levels of extracellular S1P (J), extracellular NO (K), and intracellular cGMP (L) in HUVECs transfected with siRTN3 and/or treated with ABC294640. “ns” represents p > 0.05; “*” represents p ≤ 0.05; “**” represents p ≤ 0.01; “***” represents p ≤ 0.001.

Article Snippet: Cells were seeded in 6‐well plates and transfected with RTN3 siRNA (RiboBio, Guangzhou, China) or plasmids for 24 h to operate the scraping line method, transwell, cell tube formation, and 60 μM ABC294640 (APExBIO, Houston, USA) treatment with test after 24 h, or for 48 h to operate WB, ELISA, Co‐IP, 100 μM CHX (Solarbio) treatment, and flow cytometry.

Techniques: Clinical Proteomics, Transfection, Expressing