Journal: Frontiers in Microbiology

Article Title: ProcCluster ® and procaine hydrochloride inhibit the replication of influenza A virus in vitro

doi: 10.3389/fmicb.2024.1422651

Figure Lengend Snippet: PC and PHCl inhibit PLA 2 from A549 but not MDCK cells. (A) A549 and MDCK cells were lysed for 30 min on ice using distilled water. The activity of cellular PLA 2 in the lysate was assessed in the presence of the indicated concentrations of PC and PHCl. PLA 2 activity of untreated samples was arbitrarily set to 100%. (B) Calu-3 cells were infected with IAV PR8 at an MOI of 0.1 for 30 min and then treated with the indicated concentrations of bromoenol lacton (BEL), pyrrophenone (PP) or left untreated until 24 h p.i. Virus titers were determined by standard plaque assay and the titer in the solvent-treated samples was arbitrarily set to 100%. (C) Calu-3 cells were treated (−30 min) with 30 μM bromoenol lacton (BEL) or left untreated for 30 min prior to infection. Cells were then infected with PR8 at an MOI of 1 for 30 min. Afterwards cells were treated with 30 μM BEL or solvent control (DMSO) immediately (−30 min, 30 min and DMSO) or at the indicated times (without removing the media). (A–C) Statistical significance was determined by one sample t -test comparing to 100. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Bromoenol lacton (Merck, Germany) and pyrrophenone (MedChemExpress, United States) were diluted in DMSO and stored at −20°C.

Techniques: Activity Assay, Infection, Virus, Plaque Assay, Solvent, Control

Journal: Cancer biology & therapy

Article Title: Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling.

doi: 10.1080/15384047.2019.1579961

Figure Lengend Snippet: Figure 3. Inhibition of cPLA2α inhibits the growth of cervical cancer cells. cPLA2α inhibitors pyrrophenone (a) and RSC-3388 (b) significantly inhibit cPLA2α enzyme activity. cPLA2α inhibitors pyrrophenone (c) and RSC-3388 (d) significantly inhibit proliferation of cervical cancer cells. *p < 0.05 compared to dimethyl sulfoxide (DMSO) control.

Article Snippet: Pyrrophenone and RSC-3388 were purchased from Santa Cruz Biotechnology, US.

Techniques: Inhibition, Activity Assay, Control

Journal: Cancer biology & therapy

Article Title: Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling.

doi: 10.1080/15384047.2019.1579961

Figure Lengend Snippet: Figure 4. Inhibition of cPLA2α inhibits survival without affecting migration of cervical cancer cells. Pyrrophenone (a) and RSC-3388 (b) at 100 nM significantly induce apoptosis in cervical cancer cells. Pyrrophenone (c) and RSC-3388 (d) up to 100 nM do not affect cervical cancer migration. *p < 0.05 compared to DMSO control.

Article Snippet: Pyrrophenone and RSC-3388 were purchased from Santa Cruz Biotechnology, US.

Techniques: Inhibition, Migration, Control

Journal: Cancer biology & therapy

Article Title: Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling.

doi: 10.1080/15384047.2019.1579961

Figure Lengend Snippet: Figure 5. cPLA2α inhibition using a pharmacological approach enhances chemosensitivity in cervical cancer cells. cPLA2α inhibitors pyrrophenone and RSC-3388 significantly enhance chemotherapeutic agents’ (paclitaxel and cisplatin) effects in inhibiting proliferation (a) and inducing apoptosis (b) in CaLo, SiHa, and CaSki cells. RSC-3388 and pyrrophenone at 100 nM and paclitaxel at 10 nM and cisplatin at 100 nM were used in the combination studies. *p < 0.05 compared to cPLA2α inhibitor or chemotherapeutic agent alone.

Article Snippet: Pyrrophenone and RSC-3388 were purchased from Santa Cruz Biotechnology, US.

Techniques: Inhibition

Journal: Cancer biology & therapy

Article Title: Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling.

doi: 10.1080/15384047.2019.1579961

Figure Lengend Snippet: Figure 7. cPLA2α inhibition suppresses Akt/eIF4E/β-catenin signaling in cervical cells. cPLA2α inhibition by inhibitors (a) or siRNA (b) significantly decreases cPLA2α enzyme activity in cervical cancer cells. cPLA2α inhibition by inhibitors (c) or siRNA (d) decreases βAkt (Ser473), p-eIF4E (Ser209), and β-catenin in CaLo cells. (e) Pyrrophenone and RSC-3388 at 100 nM significantly inhibit TOPflash activation. CaLo cells transfected with TOPflash plasmid were treated as indicated. The graph represents mean ± s.e.m. of TOPflash signal relative to control. cPLA2α inhibitors (f) and siRNA (g) significantly decrease the transcript level of C-MYC, BCL9, and CYCLIN D genes in CaLo cells. *p < 0.05 compared to control or siRNA con (scrambled siRNA).

Article Snippet: Pyrrophenone and RSC-3388 were purchased from Santa Cruz Biotechnology, US.

Techniques: Inhibition, Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Control

Journal: Cancer biology & therapy

Article Title: Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling.

doi: 10.1080/15384047.2019.1579961

Figure Lengend Snippet: Figure 8. cPLA2α inhibition acts on cervical cancer cells in a β-catenin-dependent manner. No significant change on β-catenin levels by pyrrophenone and RSC-3388 in CaLo cells treated with LiCl (a) or transfected with β-catenin overexpression plasmid (b). cPLA2α inhibitors (c) and siRNA (d) are ineffective in decreasing C-MYC transcript level in the presence of LiCl or β-catenin-overexpressing CaLo cells. The inhibitory effects of cPLA2α inhibition on proliferation or survival were abolished by LiCl (e and g) and β-catenin overexpression (f and h). Cells are transfected with P-Vector or P-β-catenin plasmid. Cells were harvested for Western blot, RT-PCR, and growth and apoptosis assays at 24 h post-transfection. *p < 0.05 compared to -LiCl or P-Vector.

Article Snippet: Pyrrophenone and RSC-3388 were purchased from Santa Cruz Biotechnology, US.

Techniques: Inhibition, Transfection, Over Expression, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Regulatory role of cytosolic phospholipase A 2 alpha in the induction of CD40 in microglia

doi: 10.1186/s12974-017-0811-z

Figure Lengend Snippet: cPLA 2 α activates NFκB through activation of NOX2-NADPH oxidase in the BV-2 microglia cells under LPS stimulation. a A representative immunoblot analysis of the kinetics of cPLA 2 α phosphorylation induced by 50 ng/ml LPS, out of three independent experiments. The intensity of each phosphorylated cPLA 2 α (p-cPLA 2 α Ser-505) band after quantification by densitometry was divided by the intensity of each cPLA 2 α band and expressed as arbitrary units. The bar graphs are the mean ± SE from three independent experiments. b The BV-2 cells were treated with 2 μm pyrrophenone (Pyrro) or 5 μm DPI for 60 min before stimulation with 50 ng/ml LPS for 15 min. The intensity of phosphorylated cPLA 2 α was quantitated by densitometry as described in ( a ). The bar graphs are the mean ± SE from three independent experiments. c The effect of cPLA 2 α inhibition on superoxide production in the unstimulated or stimulated BV-2 cells with 50 ng/ml LPS for 15 min was detected by DHE reduction. Two micrometer pyropheonoe (Pyrro) or 200 μm apocynin (used as a positive control) were added to the cells 60 min before stimulation with LPS. AS or sense were added 24 h prior to addition of LPS. DAPI staining shows cell nuclei. Scale bars large = 50 μm, insert = 20 μm. The intensity of reduced DHE was quantitated and expressed in the bar graph as arbitrary units. The bar graphs are the mean ± SE from three independent experiments. d Immunoprecipitation of p47 phox and phoshpo cPLA 2 α (pcPLA 2 α) in the membrane fraction of unstimulated microglia and stimulated with LPS for 15 min. Shown a representative immunoblot of three experiments. e Addition of 10 μM arachidonic acid together with LPS to cells pretreated for 24 h with antisense against cPLA 2 α restored the expression of CD40 protein. Shown a representative immunofluorescence staining of CD40. DAPI staining shows cell nuclei. The intensity of CD40 was quantitated for the cell and expressed in the bar graph as arbitrary units. Scale bars = 50 μm. The bar graph is the mean ± SE from three independent experiments. (*** p < 0.0001). f FACS analysis of CD40 protein expression in the unstimulated or stimulated BV-2 cells with 50 ng/ml LPS for 24 h in the absence or presence of 2 μm pyrrophenone or 5 μm DPI (added to the cells 60 min before stimulation with LPS). The bar graphs are the X-median ± SE from five independent experiments. g A representative immunoblot analysis of NF-κB p-65 phosphorylation (p-NFκB p-65 Ser-536) in unstimulated or stimulated BV-2 microglia by 50 ng/ml LPS for 15 min in the absence or presence of 2 μm pyrrophenone or 5 μm DPI. The intensity of each phosphorylated NF-κB p-65 band after quantification by densitometry was divided by the intensity of each NFκB p-65 band and expressed as arbitrary units. The bar graphs are the mean ± SE from three independent experiments. h A representative immunoblot analysis of phoshpo cPLA 2 α and phospho NF-κB p-65 subunit and phosspho ERK1/2 in unstimulated or stimulated BV-2 microglia by 50 ng/ml LPS for 15 min in the absence or presence of 5 μM OU126. The bar graphs are the mean ± SE of the intensity of the quantitated phosphorylated forms divided by the non-phophorylated forms of three independent experiments. i The involvement of TRIF and MyD88 pathways in activation of cPLA 2 α in the signaling leading to CD40 upregualtion. The BV-2 cells were incubated with TRIF or MyD88 peptide inhibitors for 60 min before stimulation with 50 ng/ml LPS for 15 min. A representative immunoblot analysis of cPLA 2 α activity, out of three independent experiments is presented. The intensity of phosphorylated cPLA 2 α (p-cPLA 2 α Ser-505) was quantitated by densitometry as described in A. j Shown is a representative immunofluorescence analysis of CD40 protein expression in the cells treated as in i . DAPI staining shows cell nuclei. Scale bars = 50 μm. The intensity of cPLA 2 α and of CD40 was quantitated and expressed in the bar graph as arbitrary units. The bar graph is the mean ± SE from three independent experiments. (*** p < 0.0001,** p < 0.001, n.s. not significant)

Article Snippet: Pyrrophenone was from Cayman Chemical, Michigan, USA.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Inhibition, Positive Control, Staining, Immunoprecipitation, Membrane, Expressing, Immunofluorescence, Incubation, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: Regulatory role of cytosolic phospholipase A 2 alpha in the induction of CD40 in microglia

doi: 10.1186/s12974-017-0811-z

Figure Lengend Snippet: cPLA 2 α activates NF-κB through activation of NOX2-NADPH oxidase in the BV-2 microglia cells under IFNγ stimulation. a A representative immunoblot analysis of the kinetics of cPLA 2 α phosphorylation induced by 10 ng/ml IFNγ, out of three independent experiments. The intensity of phosphorylated cPLA 2 α (p-cPLA 2 α Ser-505) was quantitated by densitometry as described in Fig. . b The BV-2 microglia cells were treated with 2 μm pyrrophenone (Pyrro) or 5 μm DPI for 1 h before stimulation with 10 ng/ml IFNγ for 4 h. Phosphorylated cPLA 2 α (p-cPLA 2 α Ser-505) intensity was quantitated by densitometry as described in Fig. . The bar graphs are the mean ± SE from three independent experiments. c The effect of cPLA 2 α inhibition on superoxide production in the unstimulated or stimulated BV-2 cells with 10 ng/ml IFNγ for 4 h detected by DHE reduction. Two micrometer pyropheonoe (Pyrro) or 200 μm apocynin (used as a positive control) were added to the cells 60 min before stimulation with IFNγ for 4 h. AS or sense (SE) were added 24 h prior to addition of IFNγ. DAPI staining shows cell nuclei. The intensity of reduced DHE was quantitated and expressed in the bar graph as arbitrary units. Scale bars large = 50 μm, insert = 20 μm. The bar graphs are the mean ± SE from three independent experiments. d Immunoprecipitation of p47 phox and phoshpo cPLA 2 α (pcPLA 2 α) in the membrane fraction of unstimulated microglia and stimulated with IFNγ for 4 h. Shown a representative immunoblot of three experiments. e Addition of 10 μM arachidonic acid togeher with IFNγ to the cells pre-treated for 24 h with antisense against cPLA 2 α restored the expression of CD40 protein. Shown a representative immunofluorescence staining of CD40. DAPI staining shows cell nuclei. The intensity of CD40 was quantitated for the cell and expressed in the bar graph as arbitrary units. Scale bars = 50 μm. f FACS analysis of CD40 protein expression in the unstimulated or stimulated BV-2 cells with10 ng/ml IFNγ for 24 h in the absence or presence of 2 μm pyrrophenone (Pyrro) or 5 μm DPI added to the cells 1 h before stimulation. The bar graphs are the X-median ± SE from five independent experiments. g A representative immunoblot analysis of phosphorylated NF-κB p65 (p-NFκB p-p65(Ser-536) in unstimulated or stimulated BV-2 microglia by 10 ng/ml IFNγ for 4 h in the absence or presence of 2 μm pyrrophenone (Pyrro) or 5 μm DPI. The intensity of each p-NFκB p-p65(Ser-536) band after quantification as described in Fig. . The bar graphs are the mean ± SE from three independent experiments. h A representative immunoblot analysis of phoshpo cPLA 2 α and phospho NF-κB p-65 subunit and phosspho ERK1/2 in unstimulated or stimulated BV-2 microglia by 10 ng/ml IFNγ for 4 h in the absence or presence of 5 μM OU126. The bar graphs are the mean ± SE of the intensity of the quantitated phosphorylated forms divided by the non-phophorylated forms of three independent experiments. (*** p < 0.0001)

Article Snippet: Pyrrophenone was from Cayman Chemical, Michigan, USA.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Inhibition, Positive Control, Staining, Immunoprecipitation, Membrane, Expressing, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Regulatory role of cytosolic phospholipase A 2 alpha in the induction of CD40 in microglia

doi: 10.1186/s12974-017-0811-z

Figure Lengend Snippet: Endogenously produced TNF-α regulates cPLA 2 α activation in the BV2 cells under IFNγ stimulation. a A representative immunoblot analysis of the kinetics of cPLA 2 α activation detected by its phosphorylated form induced by 10 ng/ml TNF-α in BV-2 microglia lysates. A representative immunoblot analysis of a dose-dependent activation of the cPLA 2 α ( b ) and of NF-kB ( c ) detected by their phosphorylated induced by TNF-α in BV-2 microglia lysates. Representative immunoblot analysis of ( d ) cPLA 2 α phosphorylation on Ser-505 and ( e ) NF-kB p-65 phosphorylation on Ser-536 in unstimulated or stimulated BV-2 microglia by 10 ng/ml IFNγ for 4 h in the absence or presence of 2 μm pyrrophenone (Pyrro) or 50 ng/ml TNF-α-neutralizing antibody (anti-TNF-α) added to the cells 60 min before stimulation with IFNγ stimulation. The intensity of each p-cPLA 2 α(Ser-505) or p-NFκB p65(Ser-536) band after quantification by densitometry was divided by the intensity of each cPLA 2 α or NFκB p65 band, respectively, and expressed as arbitrary units. The results are the mean ± SE from four experiments. f Immunofluorescence analysis of CD40 protein expression in unstimulated or stimulated BV-2 microglia with 10 ng/ml IFNγ for 24 h in the absence or presence of 50 ng/ml TNF-α-neutralizing antibody or stimulated by 10 ng/ml TNF-α. Scale bars = 50 μm. The intensity of CD40 was quantitated and expressed in the bar graph as arbitrary units. The bar graph is the mean ± SE from four independent experiments. DAPI staining shows cell nuclei. (*** p < 0.0001, n.s. not significant)

Article Snippet: Pyrrophenone was from Cayman Chemical, Michigan, USA.

Techniques: Produced, Activation Assay, Western Blot, Phospho-proteomics, Immunofluorescence, Expressing, Staining

Journal: Journal of Neuroinflammation

Article Title: Regulatory role of cytosolic phospholipase A 2 alpha in the induction of CD40 in microglia

doi: 10.1186/s12974-017-0811-z

Figure Lengend Snippet: cPLA 2 α or NADPH oxidase did not affect STAT1α activation under IFNγ stimulation. A representative immunoblot analysis of the kinetics of STAT1α phosphorylation on ( a ) tyrosine 701 or ( b ) serine 727 induced by 10 ng/ml IFNγ. A representative immunoblot analysis of STAT1α phosphorylation on ( c ) tyrosine 701 or on ( d ) serine 727 in unstimulated or stimulated BV-2 microglia by 10 ng/ml IFNγ for 15 min in the absence or presence of 2 μm pyrrophenone (Pyrro) or 5 μm DPI (added to the cells 60 min before stimulation). The intensity of each phosphorylated STAT1α (p-STAT1α) band was quantitated as described in Fig. . The Bar graphs are the mean ± SE from three experiments (*** p < 0.0001, n.s. not significant)

Article Snippet: Pyrrophenone was from Cayman Chemical, Michigan, USA.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Protein & Cell

Article Title: Proteomic analysis of ferroptosis pathways reveals a role of CEPT1 in suppressing ferroptosis

doi: 10.1093/procel/pwae004

Figure Lengend Snippet: Ferroptosis inhibition by CEPT1 is partially dependent on phospholipases. (A–D) Pulldown assays were performed with S protein beads (SFB-tagged proteins were used as the baits), and the indicated proteins were detected by WB. (E) Cell death was measured by propidium iodide (PI) staining in control (sgControl), DDHD1 -knockout, or DDHD2 -knockout (sg1/2) 786-O cells overexpressing CEPT1 or transfected with empty vector (EV) treated with 50 nmol/L RSL3 for 24 h. (F) Cell death was measured by PI staining in control (shControl) and ABHD3 -knockdown (sh1/3) 786-O cells overexpressing CEPT1 or transfected with EV treated with 50 nmol/L RSL3 for 24 h. (G and H) Cell death was measured by PI staining in control (sgControl) and PLAA -knockout (sg1/2) 786-O cells overexpressing CEPT1 or transfected with EV treated with 50 nmol/L RSL3 (G) or 10 µmol/L erastin (H) for 24 h. (I and J) Cell death was measured by PI staining in 786-O cells overexpressing CEPT1 or transfected with EV treated with DMSO, 50 nmol/L RSL3 (I), or 10 µmol/L erastin (J) with 1 µmol/L pyrrophenone (PY) or indicated combinations for 24 h. (K) PLA2 enzyme activities were measured in 786-O cells overexpressing CEPT1 WT or indicated mutants or transfected with an empty vector (EV).

Article Snippet: Ferroptosis inducer (1S,3R)-RSL3 (#19288), erastin (#17754), arachidonic acid (#90010), and pyrrophenone (#13294) were from Cayman Chemical.

Techniques: Inhibition, Staining, Control, Knock-Out, Transfection, Plasmid Preparation, Knockdown