anti ep4  (Proteintech)

Journal: Nature Communications

Article Title: Targeting prostaglandin E 2 receptor 2 in Schwann cells inhibits inflammatory pain but not inflammation

doi: 10.1038/s41467-025-63782-8

Figure Lengend Snippet: a , d , f Dose-dependent non-evoked nociception and b , e , g dose- and time-dependent allodynia after intraplantar (i.pl.) injection of PGE 2 , L-902,688 (L-902), butaprost (Buta) or vehicle (Veh) in C57BL/6 J mice (B6) ( n = 8 mice/group). c Schematic representation of agonists/antagonists targeting EP2 and EP4 receptors. h Non-evoked nociception and i allodynia after i.pl. PGE 2 (1.5 nmol) or Veh in B6 pretreated with PF-04448948 (PF, 5nmol), BGC 20-1531 (BGC, 5 nmol) or Veh ( n = 8 mice/group). j RT-qPCR for Ptger2, Ptger4 Avil and S100 mRNA in mouse dorsal root ganglia (DRG) sciatic and cutaneous Schwann cells (SCs) (DRG and sciatic SCs n = 4, cutaneous n = 3 independent experiments). k , l Representative images of EP2, EP4, NeuN and S100B expression in mouse DRG and sciatic nerve tissue (scale bar: 20 μm) ( n = 3 subjects). m Representative images of EP2, EP4 and SOX10 expression in mouse sciatic and cutaneous SCs ( n = 3 independent experiments). n–r Non-evoked nociception (left panel) and allodynia (right panel) after i.pl. PGE 2 or Veh in Plp-Cre, Adv-Cre or Control mice infected with AAV for selective silencing of EP4 (-Ptger4 ) ( P1p-Ptger4 or Adv-Ptger4 ) ( n,o ) or EP2 (-Ptger2 ) ( P1p-Ptger2 or Adv-Ptger2 ) ( p,q ) or with AAV for a scrambled shRNA ( Plp-scrambled ) ( r ) ( n = 8 mice/group). Data are mean ± s.e.m. a , d , f , h , n , o , p , q , r 1-way or b , e , g , i , n , o , p , q , r 2-way ANOVA, Bonferroni correction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. Veh † P < 0.05, †† P < 0.01, ††† P < 0.001, †††† P < 0.0001 vs. PGE 2 /Veh, Control / PGE 2 . Source data are provided as a Source Data file.

Article Snippet: Human DRGs (#0062-HP-240, Gentaur), mouse DRGs, human sciatic nerve (#0062-HP-261, Gentaur), mouse paw tissue, and mouse sciatic nerve were incubated with different primary antibodies: EP2 (#ab167171, rabbit monoclonal, Abcam, 1:250 or #APR-064, rabbit polyclonal, Alomone Labs, 1:50), EP4 (#BS-8538R, rabbit polyclonal, Bioss, 1:200), S100B (#MA1-26621, mouse monoclonal, Invitrogen, 1:50 or #ab196175, rabbit monoclonal, Abcam, 1:50), Sox10 (#AF2864, goat polyclonal, R&D Systems, 1:100), NeuN (#MAB377, mouse monoclonal, Merck, 1:250 or #MAB377X, mouse monoclonal, Merck, 1:250), GFP (#A6455, rabbit polyclonal, Invitrogen, 1:500) and glutamine synthetase (#ab64613, mouse monoclonal, Abcam, 1:1000), myelin basic protein (MBP) (#ab133620, rabbit monoclonal, Abcam, 1:500), diluted in fresh blocking solution (PBS, pH 7.4, 5% normal goat serum (NGS) or normal donkey serum (NDS)).

Techniques: Injection, Quantitative RT-PCR, Expressing, Control, Infection, shRNA

Journal: Nature Communications

Article Title: Targeting prostaglandin E 2 receptor 2 in Schwann cells inhibits inflammatory pain but not inflammation

doi: 10.1038/s41467-025-63782-8

Figure Lengend Snippet: a Global cAMP formation in human Schwann cells (hSCs), after blue light stimulation (450 nm, 10 min) ( n = 121 cells, n = 3 independent experiments) or forskolin (FSK 100 μM) ( n = 78 cells, n = 3 independent experiments). b Schematic representation of Cre recombinase dependent expression of Beggiatoa photo-activable AC (bPAC) and mCherry separated by T2A self-cleaving peptide sequence. c Representative images of mCherry expression in S100B+ cells in mouse paw tissue after intraplantar (i.pl.) infection with AAV for selective expression of bPAC in SCs (scale bar: 20 μm) ( n = 4 subjects). d Illustration of bPAC activation after blue light stimulation and allodynia induced by blue light stimulation (pulsed 1 s/5 s for 10 min) in Plp-Cre or Control mice infected with AAV- bPAC ( Plp-bPAC ) ( n = 8 mice/group). e Representative images and cumulative data of membrane and intracellular localization of EP2 and EP4 in hSCs cells, with or without pre-incubation with unlabeled PGE 2 (10 µM, 30 min). Scale bar, 20 µm. ( n = 4 independent experiments). f Concentration-dependent global cAMP formation in hSCs induced by PGE 2 ( n = 8 replicates). g Membrane confined cAMP formation induced by PGE 2 (10 nM) in hSCs in the presence of PF-04418948 (PF,1 μM), BGC 20-1531 (BGC, 1 μM) or vehicle (Veh) (cells number: PGE 2 = 80, PF = 90, BGC = 88, n = 3 independent experiments). h Illustration of membrane tagged Lyn11-bPAC membrane confined cAMP formation and cAMP formation in hSCs after blue light stimulation ( n = 47 cells, n = 3 independent experiments) or FSK (100 μM) ( n = 46 cells, n = 3 independent experiments). i Allodynia induced by blue light stimulation in Plp-Cre or Control mice infected with AAV- Lyn11-bPAC ( Plp-Lyn11-bPAC ) ( n = 8 mice/group). j Concentration-dependent PKA activation induced by PGE 2 in hSCs ( n = 8 replicates). k Membrane confined PKA activation in hSCs induced by PGE 2 (10 nM) in the presence of PF (1 μM), BGC (1 μM) or Veh (cells number: PF = 64, BGC = 63, PGE 2 = 64, n = 3 independent experiments). Data are mean ± s.e.m. a , e , h ,2-tailed Student’s t test, g , k 1-way or d , i 2-way ANOVA, Bonferroni correction. AUC area under curve. **** P < 0.0001 vs. Control/blue light. Source data are provided as a Source Data file.

Article Snippet: Human DRGs (#0062-HP-240, Gentaur), mouse DRGs, human sciatic nerve (#0062-HP-261, Gentaur), mouse paw tissue, and mouse sciatic nerve were incubated with different primary antibodies: EP2 (#ab167171, rabbit monoclonal, Abcam, 1:250 or #APR-064, rabbit polyclonal, Alomone Labs, 1:50), EP4 (#BS-8538R, rabbit polyclonal, Bioss, 1:200), S100B (#MA1-26621, mouse monoclonal, Invitrogen, 1:50 or #ab196175, rabbit monoclonal, Abcam, 1:50), Sox10 (#AF2864, goat polyclonal, R&D Systems, 1:100), NeuN (#MAB377, mouse monoclonal, Merck, 1:250 or #MAB377X, mouse monoclonal, Merck, 1:250), GFP (#A6455, rabbit polyclonal, Invitrogen, 1:500) and glutamine synthetase (#ab64613, mouse monoclonal, Abcam, 1:1000), myelin basic protein (MBP) (#ab133620, rabbit monoclonal, Abcam, 1:500), diluted in fresh blocking solution (PBS, pH 7.4, 5% normal goat serum (NGS) or normal donkey serum (NDS)).

Techniques: Expressing, Sequencing, Infection, Activation Assay, Control, Membrane, Incubation, Concentration Assay

Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: PGE2 inhibits adipogenesis through the G protein-coupled receptor EP4. (A) Schematic of IDX-induced adipogenesis in ASCs; ciliated cells begin accumulating lipids after the first 48 h of IDX treatment, and lose their cilia near the 96-h mark. Created in BioRender by Lee, M., 2025. https://BioRender.com/m4r2u0d . This figure was sublicensed under CC-BY 4.0 terms. (B) Intracellular lipid content of 3T3-L1 cells after initiation of adipogenesis using the IDX cocktail (t=0 h) in the presence of 20 µM PGE2 or DMSO vehicle during the critical first 96 h of adipogenesis. Lipid content was quantified using the integrated fluorescence intensity of BODIPY 493/503 throughout adipogenesis (left) and at the endpoint (t=144 h) (right). n =number of independent experiments. (C) Representative images showing lipid droplets in differentiated 3T3-L1 preadipocytes (t=144 h) visualized by green fluorescent BODIPY staining. Boxed areas are shown at higher magnification on the right. (D) Relative change in mRNA expression of adipogenic target genes in differentiating 3T3-L1 cells in the presence of 20 µM PGE2 or vehicle control from three independent experiments demonstrates that PGE2 inhibits adipogenic gene expression. (E,F) ASCs isolated from inguinal white adipose depots of male (E) and female (F) mice are sensitive to 20 µM PGE2 as determined by intracellular lipid content throughout adipogenesis (left) and at the endpoint (right). n =number of independent ASC isolations which pooled 2-4 mice each. (G) 3T3-L1 cells treated with the selective antagonists of EP1 (SC-19220, 10 µM), EP2 (PF-04418948, 10 µM), EP3 (L-798,106, 5 µM) and EP4 [MF498 (iEP4-1), 25 µM; MF766 (iEP4-2), 50 µM] 24 h prior to initiation of adipogenesis and then concomitant with 20 µM PGE2 for the first 48 h of adipogenesis. Dashed line marks average lipid content in control cells treated with PGE2 at endpoint. EP4 activity is required for PGE2 to inhibit adipogenesis. (H) Cas9-expressing 3T3-L1 cells, control 3T3-L1 cells expressing a safe sgRNA (sgSAFE) and EP4 knockout cells were treated with 20 µM PGE2 for the first 96 h of adipogenesis. EP4 is required for PGE2 to inhibit adipogenesis. Dashed line marks average endpoint lipid content in Cas9 control cells treated with PGE2. (I) Representative images depicting lipid droplets in cells from the data shown in H. (J) Inhibition of adipogenesis by PGE2 is recapitulated by 48 h treatment with the EP4 agonist CAY10598 (50 µM). (B-H,J) All data are mean±s.d., each data point shows an independent experiment. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 [unpaired, two-tailed Student's t -test (B,E,F) or one-way ANOVA followed by Tukey's multiple comparison test (D,G,H,J)]. a.u., arbitrary units; KO, knockout; ns, not significant.

Article Snippet: Cells were then stained for 1-4 h with antibodies targeting EP4 (Santa Cruz Biotechnology, sc-55596; 1:200), FGFR10P (Proteintech, 11343-1-AP; 1:500) and ARL13B (NeuroMabs, 75-287; 1:1000).

Techniques: Fluorescence, Staining, Expressing, Control, Gene Expression, Isolation, Activity Assay, Knock-Out, Inhibition, Two Tailed Test, Comparison

Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: EP4 localizes to primary cilia of ASCs. (A-C) Immunofluorescence staining using DAPI (blue) and antibodies recognizing the centrosomal marker FGFR1OP (magenta), the cilia marker ARL13B (red) and the EP4 receptor (green) in undifferentiated 3T3-L1 cells (A) and primary ASCs from the inguinal fat depot of male (B) and female (C) mice. Left: Representative images showing ciliated cells with enrichment of EP4 in the primary cilium. Right: The fraction of cells with primary cilia and the fraction of cells with EP4-positive cilia; n = total cell number. All cells have a nucleus and are positive for the centrosomal marker FGFR1OP, while a subset of cells have a primary cilium (ARL13B + ). (D) Whole-mount inguinal white adipose tissue stained for DAPI, ARL13B, EP4, FGFR1OP and the endothelial cell marker CD31 (magenta). Closed arrows highlight EP4 colocalization with primary cilia. (A-C) All data are mean±s.d. and each data point represents an independent experiment. Each independent primary ASC isolation pooled 2-4 mice.

Article Snippet: Cells were then stained for 1-4 h with antibodies targeting EP4 (Santa Cruz Biotechnology, sc-55596; 1:200), FGFR10P (Proteintech, 11343-1-AP; 1:500) and ARL13B (NeuroMabs, 75-287; 1:1000).

Techniques: Immunofluorescence, Staining, Marker, Isolation

Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: Ciliary EP4 is required for PGE2 to inhibit adipogenesis. (A) Cas9-expressing 3T3-L1 cells, control 3T3-L1 cells expressing a safe sgRNA (sgSAFE) and KIF3A knockout cells differentiated in the presence of 20 µM PGE2 during the first 48 h of adipogenesis. KIF3A knockout cells lacking primary cilia show no response to PGE2. (B) Cas9-expressing 3T3-L1 cells, control 3T3-L1 cells expressing a safe sgRNA, and TULP3 knockout cells differentiated in the presence of 20 µM PGE2 during the first 96 h of adipogenesis. TULP3 knockout cells lacking ciliary GPRCs show no response to PGE2. (C) Dose-dependent response of 3T3-L1 and TULP3 knockout cells to PGE2 during the first 48 h of adipogenesis. Depletion of TULP3 makes 3T3-L1 cells less sensitive to the anti-adipogenic effect of PGE2. Data are mean of three experiments ±s.d. with endpoint lipid content normalized to 3T3-L1 control cell differentiation without PGE2. EC50 calculation for 3T3-L1 and estimation for TULP3 KO performed by log (x) transformation with nonlinear fit regression. (D) 3T3-L1 cells treated with an attenuated differentiation cocktail alone or supplemented with ciliary GPCR agonist DHA and challenged with 20 µM PGE2 during the first 48 h of adipogenesis. PGE2 inhibits adipogenesis in cells treated with the cilia-dependent differentiation cocktail. Endpoint lipid accumulation was recorded after 144 h of differentiation. (A,B,D) Data are mean±s.d.; each data point represents an independent experiment. Dashed lines mark the average endpoint lipid content in control cells treated with PGE2. ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison test). a.u., arbitrary units; KO, knockout; ns, not significant.

Article Snippet: Cells were then stained for 1-4 h with antibodies targeting EP4 (Santa Cruz Biotechnology, sc-55596; 1:200), FGFR10P (Proteintech, 11343-1-AP; 1:500) and ARL13B (NeuroMabs, 75-287; 1:1000).

Techniques: Expressing, Control, Knock-Out, Cell Differentiation, Transformation Assay, Comparison

Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: Ciliary EP4 does not signal via ciliary cAMP. (A) 3T3-L1 primary cilia lengths measured at 0-, 24- and 48-h time points of adipogenesis with or without 20 µM PGE2. Unfilled circles denote lengths of individual cilia measured across four independent trials; filled circles represent the average ciliary length per trial. Mean of all four averages±s.d. shown. n =total number of cilia measured per condition across all four trials. (B) Undifferentiated 3T3-L1 cells expressing cilia-targeted cAMP biosensor treated with DMSO (black), the adenylyl cyclase activator forskolin (50 µM, blue), the FFAR4 agonist TUG891 (50 µM, green) or PGE2 (20 µM, red) at the time point indicated by the arrow; images collected every 20 s were used to calculate the ratio of fluorescence intensities between the constitutive ciliary marker and the cAMP sensor at each time point. Data are average of three independent trials ±s.e.m. n =total number of experimental wells; N =total number of cilia analyzed for each condition. (B′) Representative images of offset cAMP sensor (green) and constitutive ciliary marker (red) at the indicated time points. (C) 3T3-L1 cells treated with different PKA inhibitors (25 µM Rp-cAMPs, 25 µM Rp-8-Br-cAMPs and 10 µM H89) in the presence or absence of PGE2 during the first 96 h of adipogenesis. Dashed line marks the average endpoint lipid content in vehicle-treated control cells treated with PGE2. Only H89 rescues adipogenesis in the presence of PGE2. (D) Heat map of percentage activity remaining for different kinase targets in response to indicated kinase inhibitors. Green text denotes inhibitors that rescue adipogenesis in the presence of PGE2 (see Fig. S4E ). (E) 3T3-L1 cells differentiated in the presence or absence of 20 µM PGE2 and the kinase inhibitors H89 (10 µM) or GSK429286A (1 µM) during the first 48 h of adipogenesis. Both inhibitors rescue adipogenesis in the presence of PGE2 and inhibit ROCK2 to similar extents. (C,E) Data are mean±s.d. and each data point represents an independent experiment. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison test). a.u., arbitrary units; ns, not significant.

Article Snippet: Cells were then stained for 1-4 h with antibodies targeting EP4 (Santa Cruz Biotechnology, sc-55596; 1:200), FGFR10P (Proteintech, 11343-1-AP; 1:500) and ARL13B (NeuroMabs, 75-287; 1:1000).

Techniques: Expressing, Fluorescence, Marker, Control, Activity Assay, Comparison

Journal: Journal of Cell Science

Article Title: Prostaglandin E2 inhibits adipogenesis through the cilia-dependent activation of ROCK2

doi: 10.1242/jcs.264193

Figure Lengend Snippet: PGE2 activates ROCK2 in a cilia-dependent manner to inhibit adipogenesis. (A) 3T3-L1 cells treated with 0.5 µg/ml Rho Inhibitor I during the first 48 h partially rescues PGE2 co-treatment. Left: Endpoint lipid content with dashed line marking average lipid content of vehicle-treated control cells treated with PGE2. Right: Representative images at endpoint of adipogenesis with lipid droplets stained with BODIPY. (B) 3T3-L1 cells treated with 0.25 µg/ml Rho Activator II during the first 48 h of adipogenesis inhibits differentiation with similar efficacy as PGE2. Left: Endpoint lipid content for each condition. Right: Representative images at endpoint of adipogenesis with lipid droplets stained with BODIPY. (C) Actin network of 3T3-L1 cells visualized by phalloidin staining (red). Undifferentiated 3T3-L1 cells predominantly have actin stress fibers, which are disassembled within 48 h of adipogenesis initiation. Stress fibers are not disassembled in the presence of 20 µM PGE2, and stress fiber disassembly is rescued with ROCK2 inhibitor co-treatment (1 µM GSK429286A). Representative images of each treatment condition. Boxed areas are shown at higher magnification below. (D) Violin plot quantifying phalloidin staining intensity in 3T3-L1 cells. (E) Actin networks of TULP3 knockout cells visualized by phalloidin staining. TULP3 knockouts lacking ciliary EP4 disassemble actin stress fibers during adipogenesis regardless of PGE2 treatment or ROCK inhibition. Representative images of each treatment condition. (F) Quantification of phalloidin staining intensity in TULP3 knockout cells. (G) 20 µM PGE2 treatment does not prevent actin stress fiber disassembly in EP4 knockout cells. Representative images of each treatment condition. (H) Quantification of phalloidin staining intensity in EP4 knockout cells. (I) Model for how PGE2 inhibits adipogenesis. In wild-type 3T3-L1 cells and ASCs, activation of ciliary EP4 by PGE2 activates RhoA/ROCK2, which stabilizes the actin cytoskeleton and prevents adipogenesis. Ciliary localization of EP4 is required for this activity. Created in BioRender by Lee, M., 2025. https://BioRender.com/t962fv5 . This figure was sublicensed under CC-BY 4.0 terms. (C,E,G) Boxed areas are shown at higher magnification below. (D,F,H) Violin plots quantify phalloidin staining intensity from 18 images per condition collected equally across three independent repeats. Solid lines within each violin depict the median, while dotted lines are quartiles. Dashed lines across the graph indicate the average phalloidin staining in vehicle treated control cells after 48 h of adipogenesis. (A,B) Data are mean±s.d. and each data point represents an independent experiment. (A,B,D,F,H) * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison test). a.u., arbitrary units; ns, not significant.

Article Snippet: Cells were then stained for 1-4 h with antibodies targeting EP4 (Santa Cruz Biotechnology, sc-55596; 1:200), FGFR10P (Proteintech, 11343-1-AP; 1:500) and ARL13B (NeuroMabs, 75-287; 1:1000).

Techniques: Control, Staining, Knock-Out, Inhibition, Activation Assay, Activity Assay, Comparison