Journal: Journal of Clinical Investigation

Article Title: Inhibition of 11β–hydroxysteroid dehydrogenase type II selectively blocks the tumor COX-2 pathway and suppresses colon carcinogenesis in mice and humans

doi: 10.1172/jci37398

Figure Lengend Snippet: Figure 5 Inhibition of 11βHSD2 activity with GA suppressed tumor growth through activation of glucocorticoid receptors. (A) GA augmented low-dose CS–induced inhibition of CT26 cell COX-2 expression. (B) Treatment with 10 mg/kg/d GA i.p. led to significant decreases in tumor size, COX-2 expression, and PGE2 levels, which were completely reversed by the glucocorticoid receptor inhibitor RU486 (7.5 mg/kg/d i.m.). n = 5–6 per group. (C) GA reduced CT26 tumor expression of phosphorylated cPLA2 (P-cPLA2) and mPGES-1, but had no effect on tumor expression of COX-1 (P = 0.23) and cPGES (P = 0.055). Original magnification, ×400. (D) GA treatment significantly increased corticosterone levels and decreased 11-keto-corticosterone levels in kidney, colon, and tumors. n = 5. *P < 0.0001, **P < 0.001, ***P < 0.02, #P < 0.05, ##P < 0.01, and ###P < 0.005 versus vehicle.

Article Snippet: Affinity-purified rabbit anti-mouse 11βHSD2 (catalog no. BHSD22-A) was purchased from Alpha Diagnostic International; rabbit anti-murine COX-2 (catalog no. 160106) and COX-1 (catalog no. 160109), rabbit anti-human mPGES-1 (catalog no. 160140) and cPGES (catalog no. 160150), and rabbit anti-rat 15-PGDH (catalog no. 160615) were from Cayman Chemicals; rabbit anti-human cPLA2 (catalog no. 2382) and phosphorylated cPLA2 (Ser505; catalog no. 2831) were from Cell Signaling; and goat anti-rat VEGF (catalog no. AF564) was from R&D Systems.

Techniques: Inhibition, Activity Assay, Activation Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: Inhibition of 11β–hydroxysteroid dehydrogenase type II selectively blocks the tumor COX-2 pathway and suppresses colon carcinogenesis in mice and humans

doi: 10.1172/jci37398

Figure Lengend Snippet: Figure 7 Proposed mechanism underlying 11βHSD2 activity and colorectal tumorigenesis. In tumor cells, glucocorticoids are converted to inac- tive keto-forms by 11βHSD2, reducing glucocorticoid receptor activa- tion, while inhibition of 11βHSD2 activity (by GA and its analogs or by gene knockdown) leads to increased levels of intracellular active glucocorticoids. The consequent inhibition of cPLA2 activity and COX-2 and mPGES-1 expression results in blockade of PGE2 production and inhibition of tumor growth.

Article Snippet: Affinity-purified rabbit anti-mouse 11βHSD2 (catalog no. BHSD22-A) was purchased from Alpha Diagnostic International; rabbit anti-murine COX-2 (catalog no. 160106) and COX-1 (catalog no. 160109), rabbit anti-human mPGES-1 (catalog no. 160140) and cPGES (catalog no. 160150), and rabbit anti-rat 15-PGDH (catalog no. 160615) were from Cayman Chemicals; rabbit anti-human cPLA2 (catalog no. 2382) and phosphorylated cPLA2 (Ser505; catalog no. 2831) were from Cell Signaling; and goat anti-rat VEGF (catalog no. AF564) was from R&D Systems.

Techniques: Activity Assay, Inhibition, Knockdown, Expressing

Journal: Cancer Research

Article Title: Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells

doi: 10.1158/0008-5472.can-05-1292

Figure Lengend Snippet: Figure 3. iPLA2 and cPLA2 activities are involved in generating the chemotactic activity of peritoneal mesothelial cell conditioned medium. A, total LPA production in the conditioned medium from LP9 was inhibited by HELSS (1 Amol/L) and AACOCF3 (25 Amol/L). ***, P < 0.001 versus the corresponding control (Student’s t test). The LPA in 24 hr-CM from LP9 is f80 nmol/L/106 cells/24 hours. B, PLA2 inhibitors reduced the chemotactic activity of 24 hr-CM from LP9 on SKOV3 cell migration. When LP9 cells were incubated for conditioned medium production, AACOCF3 (25 Amol/L), HELSS (1 Amol/L), or thioether amide-PC (TAPC; 10 Amol/L) was added to the medium during the incubation for 24 hours before collecting conditioned medium (Before). We also collected conditioned medium produced in the absence of inhibitors, but added inhibitors into the lower chamber after conditioned medium were collected to assess the direct effects of these inhibitors during the migration assays (After). The effects of these conditioned medium on migration of SKOV3 cells were analyzed. Relative fold changes in migration were determined by setting cell migration in response to conditioned medium without treatment at 1.0. ***, P < 0.001 versus the control (Student’s t test). C, a to d, cell-based enzymatic activities of PLA2 in peritoneal mesothelial cells with or without overnight serum (FBS) starvation. Cells (104) were incubated with DBPC for 30 minutes. See Materials and Methods for the assay conditions. e, summary of three independent cell-based enzymatic assays. Intensities were measured as described in Materials and Methods. D, cPLA2 is constitutively phosphorylated in peritoneal mesothelial cells. Phosphorylated cPLA2 and total cPLA2 expression in peritoneal mesothelial cells were detected by corresponding antibodies in Western blot analyses. h-actin was used as a loading control. Cell lysates from LP9 and LP3 grown with or without overnight starvation from FBS were used for Western blot analyses.

Article Snippet: Cytosolic phospholipase A2 (cPLA2) antibodies were from Upstate Cell Signaling Solutions (Lake Placid, NY) or Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay, Control, Migration, Incubation, Produced, Expressing, Western Blot

Journal: Cancer Research

Article Title: Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells

doi: 10.1158/0008-5472.can-05-1292

Figure Lengend Snippet: Figure 6. Invasion of SKOV3 cells into peritoneal mesothelial cells requires LPA2, LPA3, iPLA2, cPLA2, and MEK-ERK activity. A, invasion assays were conducted in the ECIS system (see Materials and Methods). LP9 (1 105 cells in 100 AL medium) were added to the gelatin-coated wells of the ECIS chambers and allowed to form a monolayer covering the gold electrode in each well. On the next day, SKOV3, NIH3T3, or MCF7 cells (1 105 cells in 300 AL medium) were added on top of the LP9 cell layer. In the control, 300 AL medium without cells was added on top of the LP9 monolayer. B, SKOV3 cells were transiently transfected with siRNAs against LPA1-3, GAPDH, or GFP, and tested for invasion into LP9 in the ECSI system. C, for inhibitor treatments, LP9 cell monolayers were pretreated with HELSS (1 Amol/L, 30 minutes) or AACOCF3 (100 Amol/L, 30 minutes) before addition of SKOV3 cells. D, SKOV3 cells were pretreated with the MEK inhibitor PD098059 (10 Amol/L, 30 minutes; top) or an Akt inhibitor (Akti, 10 Amol/L, 30 minutes; middle) before their application to the LP9 monolayers in the ECIS wells. Bottom, SKOV3 cells were transient transfected with dominant negative (dn) MEK or Akt and tested for invasion into LP9.

Article Snippet: Cytosolic phospholipase A2 (cPLA2) antibodies were from Upstate Cell Signaling Solutions (Lake Placid, NY) or Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay, Control, Transfection, Dominant Negative Mutation

Journal: Journal of Global Health

Article Title: Unveiling the genetic biomarkers for ageing: evidence from a large sample genome-wide association study and in vivo validation

doi: 10.7189/jogh.15.04279

Figure Lengend Snippet: Panel A. Representative Nissl-stained images of the cerebral cortex and hippocampus from 3-month-old and 20-month-old mice. Panel B. Quantitative analysis of Nissl-stained images comparing the mean density of Nissl-positive signals in the cerebral cortex and hippocampus of 3m and 20m mice (n = 3). Panel C. Western blot analysis of UBA7 and PLA2G4B expression across major organs and in the brains of three- and 20-month-old mice (n = 10). Panel D. Quantification of Western blot results for UBA7 and PLA2G4B in the brains of three- and 20-month-old mice (n = 10). * P ≤ 0.05. † P ≤ 0.01. au – arbitrary units, m – month.

Article Snippet: Subsequently, we blocked the membranes using 5% skim milk and incubated them with primary antibodies targeting UBA7 (Abmart, T59622 ), PLA2G4B (Affinity, DF9429), and GAPDH (Proteintech, 10494-1-AP) overnight at 4°C.

Techniques: Staining, Western Blot, Expressing

Journal: Advanced Science

Article Title: Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation

doi: 10.1002/advs.202203995

Figure Lengend Snippet: Cytosolic phospholipase A2 (cPLA2), a nuclear mechanosensor, responds to changes in cell geometry. A) The nuclear shape index and nuclear perimeter in micropatterned SMCs were calculated. B) Representative three‐dimensional reconstruction images of the nucleus of micropatterned cells and the quantification of the nuclear surface area. Cells were infected with pSIN‐H2B‐tagGFP viruses. C) Representative fluorescent images of cPLA2‐EYFP fusion proteins in micropatterned cells. Cells were transfected with the cPLA2‐EYFP plasmids before seeding. D) Representative immunofluorescent staining of cPLA2 and nuclear envelope protein lamin B1 in micropatterned cells. Z1–Z3 indicate three different scanning layers. The distribution of cPLA2 and lamin B1 and the degree of overlap between them are indicated by fluorescence intensity profiling (right panels). E) Production of arachidonic acid (ArAc) in the micropatterned cells was assayed using ELISA. Data were obtained from eight biological repeats. In (A, B) (right panel), each dot represents a single cell. In (E), data were from eight biological repeats. Significance was assessed using a Student's t ‐test. The error bars show ± SD. The exact P values between the indicated groups are presented.

Article Snippet: To inhibit cPLA2 activation, cells were incubated with CAY10650 (MedChemExpress) at 12 nmol L −1 for 1 h. PI3K inhibitor LY294002 (Targetmol) was used at 10 μmol L −1 for 24‐h‐treatment.

Techniques: Infection, Transfection, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Single Cell

Journal: Advanced Science

Article Title: Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation

doi: 10.1002/advs.202203995

Figure Lengend Snippet: cPLA2 inhibition blocks the geometry‐regulated DNMT1 redistribution and mtDNA methylation. A) Representative immunofluorescent staining (upper panels) and quantification (lower panel) of p‐AKT in micropatterned cells treated with a cPLA2 inhibitor (CAY10650, 12 nmol L −1 ) or the control reagent (DMSO) for 1 h. B) Representative immunofluorescence staining (upper panels) and quantification (lower panel) of the nuclear/cytoplasmic ratio of DNMT1 in micropatterned cells treated with CAY10650 or DMSO. C) Representative agarose gel electrophoresis of the products of MSP for the D‐loop of bisulfite‐modified DNA isolated from micropatterned cells treated with CAY10650 or DMSO. Semi‐quantification of the MSP results is presented in the right panel. M: methylated, U: unmethylated. Data were obtained from four biological repeats. Fluorescence images were acquired using a confocal microscope. In (A, B), each dot represents a single cell. In (C), data were from four biological repeats. Significance was assessed using two‐way ANOVA with Tukey's post hoc analysis. The error bars show ± SD. The exact P values between the indicated groups are presented.

Article Snippet: To inhibit cPLA2 activation, cells were incubated with CAY10650 (MedChemExpress) at 12 nmol L −1 for 1 h. PI3K inhibitor LY294002 (Targetmol) was used at 10 μmol L −1 for 24‐h‐treatment.

Techniques: Inhibition, Methylation, Staining, Control, Immunofluorescence, Agarose Gel Electrophoresis, Modification, Isolation, Fluorescence, Microscopy, Single Cell

Journal: Advanced Science

Article Title: Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation

doi: 10.1002/advs.202203995

Figure Lengend Snippet: Intervention of the cPLA2‐AKT1‐DNMT1 pathway affects ATP production and cell contractility. A,B) Measurement of the intracellular ATP content of micropatterned SMCs subjected to the indicated treatments. Data were obtained from five biological repeats. C) Schematic representation of the MTS‐DNMT1 construct. MTS, mitochondrial targeting sequence, CMV cytomegalovirus promoter, ORF open reading frame. D) Representative immunofluorescent staining of Discosoma sp. red fluorescent protein (DsRed). Cells were transfected either with pcDNA (CL vector) or with MTS‐DNMT1. E) Representative immunofluorescence staining of DNMT1 in micropatterned cells transfected with the CL vector or MTS‐DNMT1. F) Measurement of the intracellular ATP content of micropatterned cells with pcDNA or MTS‐DNMT1 transfection. Data were obtained from five biological repeats. G) Gel contraction assay with cells with the indicated treatments. Quantifications from four biological repeats are shown in the lower panels. H) Gel contraction assay with cells subjected to DNMT1‐AA or DNMT1‐DD transfection. Quantifications from six biological repeats are shown in the lower panels. I) Traction force microscopy assay to detect the cellular contractility of micropatterned cells treated with CAY10650 (a cPLA2 inhibitor) or DMSO. Representative bright field images and cell force images are presented in the left panel. Quantification of the traction is shown in the right panel. Each dot represents a single cell. Fluorescence images were acquired using a confocal microscope. Significance was assessed using one‐way or two‐way ANOVA with Tukey's post hoc analysis. The error bars show ± SD.

Article Snippet: To inhibit cPLA2 activation, cells were incubated with CAY10650 (MedChemExpress) at 12 nmol L −1 for 1 h. PI3K inhibitor LY294002 (Targetmol) was used at 10 μmol L −1 for 24‐h‐treatment.

Techniques: Construct, Sequencing, Staining, Transfection, Plasmid Preparation, Immunofluorescence, Collagen Gel Contraction Assay, Microscopy, Single Cell, Fluorescence

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: (A) Representative cPLA2 and GAPDH Western blots in iliac arteries from early- (n = 3) versus advanced-stage (n = 3) CVD patients. (B) Scatter dot plots (arithmetic mean ± SEM) of cPLA2 protein expression normalized to GAPDH in iliac arteries from early- (n = 14) versus advanced-stage (n = 23) CVD patients. *P < 0.05 vs. early-stage CVD group (unpaired Mann-Whitney test).

Article Snippet: For human, histologic paraffin sections of iliac arteries samples were cut at 2 μm, mounted on charged slides, and stained with H&E and von Kossa for pathological evaluation. cPLA2 staining was performed on 1–2 μm paraffin sections of A . ilia communis samples of histopathologically classified early ( n = 15) and advanced ( n = 24) patients, using a 1:50 dilution of the rabbit anti-human PLA2G4A antibody (ProSci Incorporated).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: (A) Scatter dot plots (arithmetic mean ± SEM) (n = 5–6, μg/mg protein) of calcium content in aortic tissue of mice receiving vehicle (CTR) or high-dosed cholecalciferol (vD), without or with additional AACOCF3 (AAC) treatment. (B) Scatter dot plots (arithmetic mean ± SEM) (n = 5–6; arbitrary units [a.u.]) of Pla2g4a relative mRNA expression in aortic tissue of mice receiving vehicle or high-dosed cholecalciferol, without or with additional AACOCF3 treatment. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice; †P < 0.05 vs. vD-treated mice (1-way ANOVA with Tukey-HSD post-hoc test).

Article Snippet: For human, histologic paraffin sections of iliac arteries samples were cut at 2 μm, mounted on charged slides, and stained with H&E and von Kossa for pathological evaluation. cPLA2 staining was performed on 1–2 μm paraffin sections of A . ilia communis samples of histopathologically classified early ( n = 15) and advanced ( n = 24) patients, using a 1:50 dilution of the rabbit anti-human PLA2G4A antibody (ProSci Incorporated).

Techniques: Expressing, Control

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: Scatter dot plots (arithmetic mean ± SEM) (n = 6, arbitrary units [a.u.]) of PLA2G4A (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Neg.si-silenced HAoSMCs; †P < 0.05, †††P < 0.001 vs. Neg.si-silenced and Pi-treated HAoSMCs (1-way ANOVA with Games-Howell post-hoc test for A or with Tukey-HSD post-hoc test for C and Steel-Dwass method for B and D).

Article Snippet: For human, histologic paraffin sections of iliac arteries samples were cut at 2 μm, mounted on charged slides, and stained with H&E and von Kossa for pathological evaluation. cPLA2 staining was performed on 1–2 μm paraffin sections of A . ilia communis samples of histopathologically classified early ( n = 15) and advanced ( n = 24) patients, using a 1:50 dilution of the rabbit anti-human PLA2G4A antibody (ProSci Incorporated).

Techniques: Expressing, Negative Control