Review

sphk2 inhibitor abc294640 (Echelon Biosciences)

Name: sphk2 inhibitor abc294640
Brand: Echelon Biosciences
Rating: 93 (4 reviews)

Echelon Biosciences is a verified supplier

Echelon Biosciences manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Echelon Biosciences sphk2 inhibitor abc294640
$Endogenous AHR and <t>SPHK2</t> interact in HeLa cells. A,B) HeLa cells treated with or without TCDF were subjected to cytoplasmic/nuclear extract fractionation and immunoprecipitated using AHR antibody and western blotting analysis was performed using an (A) AHR antibody or (B) SPHK2 antibody. C, Control; T, TCDF treated samples. C) After IP of SPHK2, western blotting analysis was performed with AHR, ARNT and SPHK2 antibodies. C, Control; T, TCDF treated samples. D) After IP of ARNT, western blotting analysis was performed with AHR, SPHK2 and ARNT antibodies. C, Control; T, TCDF treated samples. E) Immunofluorescent double staining using anti‐AHR and anti‐SPHK2 antibodies. The nucleus was counterstained with Hoechst 33 342. Arrows c indicate the cytoplasm, n indicate the nucleus. Bar is 10 µm. Data are representative images from three independent experiments.$
Sphk2 Inhibitor Abc294640, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sphk2 inhibitor abc294640/product/Echelon Biosciences
Average 93 stars, based on 1 article reviews

sphk2 inhibitor abc294640 - by Bioz Stars, 2025-04

93/100 stars

Images

1) Product Images from "Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation"

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

Journal: Advanced Science

doi: 10.1002/advs.202400794

$Endogenous AHR and SPHK2 interact in HeLa cells. A,B) HeLa cells treated with or without TCDF were subjected to cytoplasmic/nuclear extract fractionation and immunoprecipitated using AHR antibody and western blotting analysis was performed using an (A) AHR antibody or (B) SPHK2 antibody. C, Control; T, TCDF treated samples. C) After IP of SPHK2, western blotting analysis was performed with AHR, ARNT and SPHK2 antibodies. C, Control; T, TCDF treated samples. D) After IP of ARNT, western blotting analysis was performed with AHR, SPHK2 and ARNT antibodies. C, Control; T, TCDF treated samples. E) Immunofluorescent double staining using anti‐AHR and anti‐SPHK2 antibodies. The nucleus was counterstained with Hoechst 33 342. Arrows c indicate the cytoplasm, n indicate the nucleus. Bar is 10 µm. Data are representative images from three independent experiments.$
Figure Legend Snippet: Endogenous AHR and SPHK2 interact in HeLa cells. A,B) HeLa cells treated with or without TCDF were subjected to cytoplasmic/nuclear extract fractionation and immunoprecipitated using AHR antibody and western blotting analysis was performed using an (A) AHR antibody or (B) SPHK2 antibody. C, Control; T, TCDF treated samples. C) After IP of SPHK2, western blotting analysis was performed with AHR, ARNT and SPHK2 antibodies. C, Control; T, TCDF treated samples. D) After IP of ARNT, western blotting analysis was performed with AHR, SPHK2 and ARNT antibodies. C, Control; T, TCDF treated samples. E) Immunofluorescent double staining using anti‐AHR and anti‐SPHK2 antibodies. The nucleus was counterstained with Hoechst 33 342. Arrows c indicate the cytoplasm, n indicate the nucleus. Bar is 10 µm. Data are representative images from three independent experiments.

Techniques Used: Fractionation, Immunoprecipitation, Western Blot, Control, Double Staining

$Knockdown of SPHK2 downregulates AHR expression. A) qRT‐PCR analysis of CYP1A1 expression in HeLa cells after incubation with or without TCDF (0.1 µM) for 2 h or pretreatment with SPHK2 inhibitor (ABC, ABC294640) for 2 h. Carrier solvent DMSO was used as a control. B) CCK8 assay results after 2 h incubation with different doses of ABC294640 (µM). C) HeLa cells after treatment with 0.1 µM TCDF for 30 min or pretreatment with ABC294640 for 2 h followed by 0.1 µM TCDF for 30 min, cells were fractionated into cytoplasmic and nuclear extracts and subjected to western blotting analysis with the indicated antibodies. Red arrow indicates the reduced level of AHR protein in the ABC294640 + TCDF treated nucleus. LAMIN A/C antibody and GAPDH antibody were used for the loading control of nuclear and cytoplasmic extracts, respectively. n = 3 D) HeLa cells were transfected with SPHK2 shRNA for 48 h and the efficiency of each shRNA plasmid knockdown was assessed by qRT‐PCR (left) and western blotting (right). n = 3 E) Western blot analysis of HeLa cells transfected with SPHK2 shRNA or shRNA‐scramble (scr) for 48 h. Cells were treated 30 min with or without 0.1 µM TCDF, extracted, and proteins detected with the indicated antibodies. n = 3 F) ABC294640 incubation in HeLa cells for 2 h decreased AHR expression as determined by western blotting analysis. n = 3 G) shRNA for SPHK2 for 48 h decreases AHR mRNA in HeLa cells. (H) Transient overexpression of human SPHK2 for 48 h and qRT‐PCR was performed. All data mean ± S.D. (n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.$
Figure Legend Snippet: Knockdown of SPHK2 downregulates AHR expression. A) qRT‐PCR analysis of CYP1A1 expression in HeLa cells after incubation with or without TCDF (0.1 µM) for 2 h or pretreatment with SPHK2 inhibitor (ABC, ABC294640) for 2 h. Carrier solvent DMSO was used as a control. B) CCK8 assay results after 2 h incubation with different doses of ABC294640 (µM). C) HeLa cells after treatment with 0.1 µM TCDF for 30 min or pretreatment with ABC294640 for 2 h followed by 0.1 µM TCDF for 30 min, cells were fractionated into cytoplasmic and nuclear extracts and subjected to western blotting analysis with the indicated antibodies. Red arrow indicates the reduced level of AHR protein in the ABC294640 + TCDF treated nucleus. LAMIN A/C antibody and GAPDH antibody were used for the loading control of nuclear and cytoplasmic extracts, respectively. n = 3 D) HeLa cells were transfected with SPHK2 shRNA for 48 h and the efficiency of each shRNA plasmid knockdown was assessed by qRT‐PCR (left) and western blotting (right). n = 3 E) Western blot analysis of HeLa cells transfected with SPHK2 shRNA or shRNA‐scramble (scr) for 48 h. Cells were treated 30 min with or without 0.1 µM TCDF, extracted, and proteins detected with the indicated antibodies. n = 3 F) ABC294640 incubation in HeLa cells for 2 h decreased AHR expression as determined by western blotting analysis. n = 3 G) shRNA for SPHK2 for 48 h decreases AHR mRNA in HeLa cells. (H) Transient overexpression of human SPHK2 for 48 h and qRT‐PCR was performed. All data mean ± S.D. (n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Techniques Used: Knockdown, Expressing, Quantitative RT-PCR, Incubation, Solvent, Control, CCK-8 Assay, Western Blot, Transfection, shRNA, Plasmid Preparation, Over Expression

AHR agonist activates the SPHK2/S1P pathway resulting in rapid nuclear retention of the AHR and SPHK2. A) Immunofluorescent images of HeLa cells for p‐SPHK2 levels after TCDF (0.1 µM) incubation for 20 min. Arrowheads indicate the peri‐nuclear region staining of p‐SPHK2 in control cells and arrows indicate the nuclear expression of p‐SPHK2 in TCDF treated cells. Hoechst 33342 was used for the counter staining of nuclei. Bar = 20 µm. Data are representative images from three independent experiments. B) Western blot analysis of HeLa cells after TCDF treatment for 15 min. C, Control; T, TCDF (n = 3). C) qRT‐PCR analysis of HeLa cells for AHR and SPHK2 mRNA levels after S1P incubation for 30 min. C: Control. Data are mean ± S.D. (n = 3). **** p < 0.0001, * p < 0.05. D) Western blotting analysis of HeLa cells after S1P treatment for 5 min. Cytoplasmic and nuclear extracts were obtained and western blotting analysis conducted with the antibodies indicated (n = 3). E) Western blotting analysis of HeLa cell extracts after 5‐ and 10‐min treatment with S1P (10 µM). GAPDH and histone H3 (H3) antibodies were used as internal controls for cytoplasmic and nuclear extracts, respectively. All data are representative images of three independent experiments (n = 3). F) Pulldown assays using S1P immobilized on agarose beads or equivalent control beads. After extensive washing, bound proteins were dissolved in SDS sample buffer and separated by SDS‐PAGE and subjected to western blotting analysis (n = 3). C, Control; T, TCDF treated, respectively. Images are representative of three independent experiments.

Figure Legend Snippet: AHR agonist activates the SPHK2/S1P pathway resulting in rapid nuclear retention of the AHR and SPHK2. A) Immunofluorescent images of HeLa cells for p‐SPHK2 levels after TCDF (0.1 µM) incubation for 20 min. Arrowheads indicate the peri‐nuclear region staining of p‐SPHK2 in control cells and arrows indicate the nuclear expression of p‐SPHK2 in TCDF treated cells. Hoechst 33342 was used for the counter staining of nuclei. Bar = 20 µm. Data are representative images from three independent experiments. B) Western blot analysis of HeLa cells after TCDF treatment for 15 min. C, Control; T, TCDF (n = 3). C) qRT‐PCR analysis of HeLa cells for AHR and SPHK2 mRNA levels after S1P incubation for 30 min. C: Control. Data are mean ± S.D. (n = 3). **** p < 0.0001, * p < 0.05. D) Western blotting analysis of HeLa cells after S1P treatment for 5 min. Cytoplasmic and nuclear extracts were obtained and western blotting analysis conducted with the antibodies indicated (n = 3). E) Western blotting analysis of HeLa cell extracts after 5‐ and 10‐min treatment with S1P (10 µM). GAPDH and histone H3 (H3) antibodies were used as internal controls for cytoplasmic and nuclear extracts, respectively. All data are representative images of three independent experiments (n = 3). F) Pulldown assays using S1P immobilized on agarose beads or equivalent control beads. After extensive washing, bound proteins were dissolved in SDS sample buffer and separated by SDS‐PAGE and subjected to western blotting analysis (n = 3). C, Control; T, TCDF treated, respectively. Images are representative of three independent experiments.

Techniques Used: Incubation, Staining, Control, Expressing, Western Blot, Quantitative RT-PCR, SDS Page

Characterization of SPHK2 and AHR occupancy on the CYP1A1 promoter. A) ChIP‐qRT‐PCR analysis for the presence of the AHR in the proximity of the two DRE in the CYP1A1 promoter in HeLa cells treated with or without 0.1 µM TCDF for 30 min or SPHK2 inhibitor ABC294640 (30 µM) pretreatment for 3 h followed by TCDF treatment for 30 min. Normal rabbit IgG was used for the negative control. C, control; T, TCDF; ABC, ABC294640. B) ChIP‐qPCR analysis for AHR and SPHK2 occupancy on the CYP1A1 promoter in HeLa cells treated with or without TCDF (0.1 µM) for 5 min. C, control; T, TCDF. C) ChIP‐qPCR analysis for AHR occupancy proximal to each DRE in the CYP1A1 promoter in HeLa cells with or without TCDF (0.1 µM) or S1P (1 µM) for 5 min. D) ChIP‐qPCR analysis for AHR occupancy proximal to each DRE in the CYP1A1 promoter in HeLa cells with or without S1P (1 µM) for 5, 30, 60 min. All data are representative of independent three experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01. * p < 0.05.

Figure Legend Snippet: Characterization of SPHK2 and AHR occupancy on the CYP1A1 promoter. A) ChIP‐qRT‐PCR analysis for the presence of the AHR in the proximity of the two DRE in the CYP1A1 promoter in HeLa cells treated with or without 0.1 µM TCDF for 30 min or SPHK2 inhibitor ABC294640 (30 µM) pretreatment for 3 h followed by TCDF treatment for 30 min. Normal rabbit IgG was used for the negative control. C, control; T, TCDF; ABC, ABC294640. B) ChIP‐qPCR analysis for AHR and SPHK2 occupancy on the CYP1A1 promoter in HeLa cells treated with or without TCDF (0.1 µM) for 5 min. C, control; T, TCDF. C) ChIP‐qPCR analysis for AHR occupancy proximal to each DRE in the CYP1A1 promoter in HeLa cells with or without TCDF (0.1 µM) or S1P (1 µM) for 5 min. D) ChIP‐qPCR analysis for AHR occupancy proximal to each DRE in the CYP1A1 promoter in HeLa cells with or without S1P (1 µM) for 5, 30, 60 min. All data are representative of independent three experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01. * p < 0.05.

Techniques Used: Quantitative RT-PCR, Negative Control, Control

$S1P rescues the downregulation of CYP1A1 in CRSPR/Cas9‐mediated SPHK2‐KO cells. A) Western blotting analysis of HeLa CRSPR/Cas9‐treated cells after 30 min treatment with/without TCDF (0.1 µM), WT control or SPHK2‐KO HeLa cells were fractionated to isolate cytoplasmic and nuclear extracts and protein expression was assessed using antibodies as indicated. C, Control; T, TCDF treated, respectively. B) qPCR analysis of CYP1A1 expression in WT control HeLa cells or SPHK2‐KO HeLa cells 2 h after dose‐dependent TCDF exposure. C) qPCR analysis for CYP1A1 expression in WT control HeLa cells, SPHK2‐KO HeLa cells, and SPHK2‐KO+S1P HeLa cells. Cells treated with TCDF (0.1 and 1 nM). D) ChIP‐qPCR analysis for the presence of the AHR in the two DRE in the CYP1A1 promoter in WT control or SPHK2‐KO HeLa cells treated with or without 1 µM S1P for 5 min. Normal rabbit IgG was used for the negative control. C, control; T, TCDF; ABC, ABC294640; 2KO, SPHK2‐KO. All data are representative of three independent experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01. * p < 0.05.$
Figure Legend Snippet: S1P rescues the downregulation of CYP1A1 in CRSPR/Cas9‐mediated SPHK2‐KO cells. A) Western blotting analysis of HeLa CRSPR/Cas9‐treated cells after 30 min treatment with/without TCDF (0.1 µM), WT control or SPHK2‐KO HeLa cells were fractionated to isolate cytoplasmic and nuclear extracts and protein expression was assessed using antibodies as indicated. C, Control; T, TCDF treated, respectively. B) qPCR analysis of CYP1A1 expression in WT control HeLa cells or SPHK2‐KO HeLa cells 2 h after dose‐dependent TCDF exposure. C) qPCR analysis for CYP1A1 expression in WT control HeLa cells, SPHK2‐KO HeLa cells, and SPHK2‐KO+S1P HeLa cells. Cells treated with TCDF (0.1 and 1 nM). D) ChIP‐qPCR analysis for the presence of the AHR in the two DRE in the CYP1A1 promoter in WT control or SPHK2‐KO HeLa cells treated with or without 1 µM S1P for 5 min. Normal rabbit IgG was used for the negative control. C, control; T, TCDF; ABC, ABC294640; 2KO, SPHK2‐KO. All data are representative of three independent experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01. * p < 0.05.

Techniques Used: Western Blot, Control, Expressing, Negative Control

The LXXLL motif facilitates the binding of SPHK2 to AHR in the nucleus. A) Alignment of LXXLL motif sequences present in human and mouse SPHK2. B) (Top)Scheme of site‐directed mutagenesis of LXXLL motif. Three of the human SPHK2 LXXLLs motif are indicated. Numbering indicates the position of the amino acid substitution. (Bottom) Co‐IP image of nuclear extracts of COS‐1 cells that were transfected with hAHR‐FLAG and hSPHK2‐HA WT or LXXLL single, double, and triple mutation plasmids for 48 h followed by TCDF treatment for 30 min. C) (Top)Scheme of site‐directed mutagenesis of LXXLL motif. (Bottom) Co‐IP image of cytoplasm and nuclear extracts of COS‐1 cells after transfection with both hAHR‐FLAG and hSPHK2‐HA WT or SPHK2‐LXXLL single mutation plasmids for 48 h followed by TCDF treatment for 30 min. V, control vector. D) Immunofluorescence images of COS‐1 cells co‐transfected with AHR‐FLAG and either SPHK2‐WT, SPHK2‐NLS deletion (NLSΔ), or SPHK2‐triple LXXLL mutation plasmids (LXXLL‐m). (Bottom) Quantitative analysis of the percentage of AHR+ nuclear per SPHK2 + cells in randomly selected independent fields. n > 200 cells (fields n > 5). E) Immunofluorescence images of SPHK2‐KO HeLa cells transfected with control vector (Vector), SPHK2‐WT, SPHK2‐NLS deletion (NLSΔ), or SPHK2‐triple LXXLL mutation plasmids (LXXLL‐m). (Bottom) Quantitative analysis of percentage of AHR+ nuclear staining per SPHK2 + cells in randomly selected independent fields. n > 200 (fields n > 13). Bar = 20 um.

Figure Legend Snippet: The LXXLL motif facilitates the binding of SPHK2 to AHR in the nucleus. A) Alignment of LXXLL motif sequences present in human and mouse SPHK2. B) (Top)Scheme of site‐directed mutagenesis of LXXLL motif. Three of the human SPHK2 LXXLLs motif are indicated. Numbering indicates the position of the amino acid substitution. (Bottom) Co‐IP image of nuclear extracts of COS‐1 cells that were transfected with hAHR‐FLAG and hSPHK2‐HA WT or LXXLL single, double, and triple mutation plasmids for 48 h followed by TCDF treatment for 30 min. C) (Top)Scheme of site‐directed mutagenesis of LXXLL motif. (Bottom) Co‐IP image of cytoplasm and nuclear extracts of COS‐1 cells after transfection with both hAHR‐FLAG and hSPHK2‐HA WT or SPHK2‐LXXLL single mutation plasmids for 48 h followed by TCDF treatment for 30 min. V, control vector. D) Immunofluorescence images of COS‐1 cells co‐transfected with AHR‐FLAG and either SPHK2‐WT, SPHK2‐NLS deletion (NLSΔ), or SPHK2‐triple LXXLL mutation plasmids (LXXLL‐m). (Bottom) Quantitative analysis of the percentage of AHR+ nuclear per SPHK2 + cells in randomly selected independent fields. n > 200 cells (fields n > 5). E) Immunofluorescence images of SPHK2‐KO HeLa cells transfected with control vector (Vector), SPHK2‐WT, SPHK2‐NLS deletion (NLSΔ), or SPHK2‐triple LXXLL mutation plasmids (LXXLL‐m). (Bottom) Quantitative analysis of percentage of AHR+ nuclear staining per SPHK2 + cells in randomly selected independent fields. n > 200 (fields n > 13). Bar = 20 um.

Techniques Used: Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining

Schematic illustration of the dual function of SPHK2. A) SPHK2 functions as a cofactor for the AHR/ARNT heterodimer on the DRE‐containing promoter region of the CYP1A1 gene. B) SPHK2, S1P, and AHR establish a positive feedback mechanism for ceramide de novo biosynthesis metabolism. S1P also enhances AHR recruitment to DREs.

Figure Legend Snippet: Schematic illustration of the dual function of SPHK2. A) SPHK2 functions as a cofactor for the AHR/ARNT heterodimer on the DRE‐containing promoter region of the CYP1A1 gene. B) SPHK2, S1P, and AHR establish a positive feedback mechanism for ceramide de novo biosynthesis metabolism. S1P also enhances AHR recruitment to DREs.

Techniques Used:

Image Search Results

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: Endogenous AHR and SPHK2 interact in HeLa cells. A,B) HeLa cells treated with or without TCDF were subjected to cytoplasmic/nuclear extract fractionation and immunoprecipitated using AHR antibody and western blotting analysis was performed using an (A) AHR antibody or (B) SPHK2 antibody. C, Control; T, TCDF treated samples. C) After IP of SPHK2, western blotting analysis was performed with AHR, ARNT and SPHK2 antibodies. C, Control; T, TCDF treated samples. D) After IP of ARNT, western blotting analysis was performed with AHR, SPHK2 and ARNT antibodies. C, Control; T, TCDF treated samples. E) Immunofluorescent double staining using anti‐AHR and anti‐SPHK2 antibodies. The nucleus was counterstained with Hoechst 33 342. Arrows c indicate the cytoplasm, n indicate the nucleus. Bar is 10 µm. Data are representative images from three independent experiments.

Article Snippet: Main reagents used in this manuscript were as follows; 2,3,7,8‐Tetrachlorodibenzofuran (TCDF, #EF‐903‐C, Cambridge Isotope Laboratories), myriocin (from Mycelia sterilia, #M1177, SIGMA), S1P bioactive lipid (#73 914, SIGMA), SPHK2 inhibitor ABC294640 (C 23 H 25 ClN 2 O, #B‐0025, Echelon Biosciences Inc.).

Techniques: Fractionation, Immunoprecipitation, Western Blot, Control, Double Staining

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: Knockdown of SPHK2 downregulates AHR expression. A) qRT‐PCR analysis of CYP1A1 expression in HeLa cells after incubation with or without TCDF (0.1 µM) for 2 h or pretreatment with SPHK2 inhibitor (ABC, ABC294640) for 2 h. Carrier solvent DMSO was used as a control. B) CCK8 assay results after 2 h incubation with different doses of ABC294640 (µM). C) HeLa cells after treatment with 0.1 µM TCDF for 30 min or pretreatment with ABC294640 for 2 h followed by 0.1 µM TCDF for 30 min, cells were fractionated into cytoplasmic and nuclear extracts and subjected to western blotting analysis with the indicated antibodies. Red arrow indicates the reduced level of AHR protein in the ABC294640 + TCDF treated nucleus. LAMIN A/C antibody and GAPDH antibody were used for the loading control of nuclear and cytoplasmic extracts, respectively. n = 3 D) HeLa cells were transfected with SPHK2 shRNA for 48 h and the efficiency of each shRNA plasmid knockdown was assessed by qRT‐PCR (left) and western blotting (right). n = 3 E) Western blot analysis of HeLa cells transfected with SPHK2 shRNA or shRNA‐scramble (scr) for 48 h. Cells were treated 30 min with or without 0.1 µM TCDF, extracted, and proteins detected with the indicated antibodies. n = 3 F) ABC294640 incubation in HeLa cells for 2 h decreased AHR expression as determined by western blotting analysis. n = 3 G) shRNA for SPHK2 for 48 h decreases AHR mRNA in HeLa cells. (H) Transient overexpression of human SPHK2 for 48 h and qRT‐PCR was performed. All data mean ± S.D. (n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Incubation, Solvent, Control, CCK-8 Assay, Western Blot, Transfection, shRNA, Plasmid Preparation, Over Expression

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: AHR agonist activates the SPHK2/S1P pathway resulting in rapid nuclear retention of the AHR and SPHK2. A) Immunofluorescent images of HeLa cells for p‐SPHK2 levels after TCDF (0.1 µM) incubation for 20 min. Arrowheads indicate the peri‐nuclear region staining of p‐SPHK2 in control cells and arrows indicate the nuclear expression of p‐SPHK2 in TCDF treated cells. Hoechst 33342 was used for the counter staining of nuclei. Bar = 20 µm. Data are representative images from three independent experiments. B) Western blot analysis of HeLa cells after TCDF treatment for 15 min. C, Control; T, TCDF (n = 3). C) qRT‐PCR analysis of HeLa cells for AHR and SPHK2 mRNA levels after S1P incubation for 30 min. C: Control. Data are mean ± S.D. (n = 3). **** p < 0.0001, * p < 0.05. D) Western blotting analysis of HeLa cells after S1P treatment for 5 min. Cytoplasmic and nuclear extracts were obtained and western blotting analysis conducted with the antibodies indicated (n = 3). E) Western blotting analysis of HeLa cell extracts after 5‐ and 10‐min treatment with S1P (10 µM). GAPDH and histone H3 (H3) antibodies were used as internal controls for cytoplasmic and nuclear extracts, respectively. All data are representative images of three independent experiments (n = 3). F) Pulldown assays using S1P immobilized on agarose beads or equivalent control beads. After extensive washing, bound proteins were dissolved in SDS sample buffer and separated by SDS‐PAGE and subjected to western blotting analysis (n = 3). C, Control; T, TCDF treated, respectively. Images are representative of three independent experiments.

Techniques: Incubation, Staining, Control, Expressing, Western Blot, Quantitative RT-PCR, SDS Page

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: Characterization of SPHK2 and AHR occupancy on the CYP1A1 promoter. A) ChIP‐qRT‐PCR analysis for the presence of the AHR in the proximity of the two DRE in the CYP1A1 promoter in HeLa cells treated with or without 0.1 µM TCDF for 30 min or SPHK2 inhibitor ABC294640 (30 µM) pretreatment for 3 h followed by TCDF treatment for 30 min. Normal rabbit IgG was used for the negative control. C, control; T, TCDF; ABC, ABC294640. B) ChIP‐qPCR analysis for AHR and SPHK2 occupancy on the CYP1A1 promoter in HeLa cells treated with or without TCDF (0.1 µM) for 5 min. C, control; T, TCDF. C) ChIP‐qPCR analysis for AHR occupancy proximal to each DRE in the CYP1A1 promoter in HeLa cells with or without TCDF (0.1 µM) or S1P (1 µM) for 5 min. D) ChIP‐qPCR analysis for AHR occupancy proximal to each DRE in the CYP1A1 promoter in HeLa cells with or without S1P (1 µM) for 5, 30, 60 min. All data are representative of independent three experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01. * p < 0.05.

Techniques: Quantitative RT-PCR, Negative Control, Control

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: S1P rescues the downregulation of CYP1A1 in CRSPR/Cas9‐mediated SPHK2‐KO cells. A) Western blotting analysis of HeLa CRSPR/Cas9‐treated cells after 30 min treatment with/without TCDF (0.1 µM), WT control or SPHK2‐KO HeLa cells were fractionated to isolate cytoplasmic and nuclear extracts and protein expression was assessed using antibodies as indicated. C, Control; T, TCDF treated, respectively. B) qPCR analysis of CYP1A1 expression in WT control HeLa cells or SPHK2‐KO HeLa cells 2 h after dose‐dependent TCDF exposure. C) qPCR analysis for CYP1A1 expression in WT control HeLa cells, SPHK2‐KO HeLa cells, and SPHK2‐KO+S1P HeLa cells. Cells treated with TCDF (0.1 and 1 nM). D) ChIP‐qPCR analysis for the presence of the AHR in the two DRE in the CYP1A1 promoter in WT control or SPHK2‐KO HeLa cells treated with or without 1 µM S1P for 5 min. Normal rabbit IgG was used for the negative control. C, control; T, TCDF; ABC, ABC294640; 2KO, SPHK2‐KO. All data are representative of three independent experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01. * p < 0.05.

Techniques: Western Blot, Control, Expressing, Negative Control

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: The LXXLL motif facilitates the binding of SPHK2 to AHR in the nucleus. A) Alignment of LXXLL motif sequences present in human and mouse SPHK2. B) (Top)Scheme of site‐directed mutagenesis of LXXLL motif. Three of the human SPHK2 LXXLLs motif are indicated. Numbering indicates the position of the amino acid substitution. (Bottom) Co‐IP image of nuclear extracts of COS‐1 cells that were transfected with hAHR‐FLAG and hSPHK2‐HA WT or LXXLL single, double, and triple mutation plasmids for 48 h followed by TCDF treatment for 30 min. C) (Top)Scheme of site‐directed mutagenesis of LXXLL motif. (Bottom) Co‐IP image of cytoplasm and nuclear extracts of COS‐1 cells after transfection with both hAHR‐FLAG and hSPHK2‐HA WT or SPHK2‐LXXLL single mutation plasmids for 48 h followed by TCDF treatment for 30 min. V, control vector. D) Immunofluorescence images of COS‐1 cells co‐transfected with AHR‐FLAG and either SPHK2‐WT, SPHK2‐NLS deletion (NLSΔ), or SPHK2‐triple LXXLL mutation plasmids (LXXLL‐m). (Bottom) Quantitative analysis of the percentage of AHR+ nuclear per SPHK2 + cells in randomly selected independent fields. n > 200 cells (fields n > 5). E) Immunofluorescence images of SPHK2‐KO HeLa cells transfected with control vector (Vector), SPHK2‐WT, SPHK2‐NLS deletion (NLSΔ), or SPHK2‐triple LXXLL mutation plasmids (LXXLL‐m). (Bottom) Quantitative analysis of percentage of AHR+ nuclear staining per SPHK2 + cells in randomly selected independent fields. n > 200 (fields n > 13). Bar = 20 um.

Techniques: Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation

doi: 10.1002/advs.202400794

Figure Lengend Snippet: Schematic illustration of the dual function of SPHK2. A) SPHK2 functions as a cofactor for the AHR/ARNT heterodimer on the DRE‐containing promoter region of the CYP1A1 gene. B) SPHK2, S1P, and AHR establish a positive feedback mechanism for ceramide de novo biosynthesis metabolism. S1P also enhances AHR recruitment to DREs.

Techniques:

(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.

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

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.

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.

Techniques: Control, Staining, Western Blot, Quantitation Assay

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

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.

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

(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.

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.

Techniques: Dot Blot, Expressing, Western Blot, Transfection

(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.

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.

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

(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.

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.

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

(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.

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.

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

Journal: Cell reports

Article Title: Neurons with Complex Karyotypes Are Rare in Aged Human Neocortex

doi: 10.1016/j.celrep.2018.12.107

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Dounce Tissue grinder Pestle A clearance 0.0030–0.0050 in. Pestle B clearance 0.0005–0.0025 in. , Kimble via Sigma-Aldrich , Cat. # D8938.

Techniques: Recombinant, Protease Inhibitor, Staining, Purification, Sequencing, Software

Review

Structured Review

Images

1) Product Images from "Sphingosine Kinase 2 Regulates Aryl Hydrocarbon Receptor Nuclear Translocation and Target Gene Activation"

Similar Products

Image Search Results