pmx205  (Tocris)

Journal: bioRxiv

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment by regulating tumour cell fate

doi: 10.1101/2024.09.27.615431

Figure Lengend Snippet: For the whole figure: Individual biological replicates (large points) represent the average of the technical replicates (small points) . p values were calculated using biological replicates (large points) by one-way ANOVA with Dunnett’s test (A and B), two-way ANOVA with uncorrected Fisher’s LSD test (C, I and J), and two-tailed paired Student’s t test (F and H). (A-B) HCT116 cells were pretreated for 8 hr with the indicated dose of C5aR1 antagonists, PMX205, JPE-1375 and Avacopan. Cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 40 hr and subjected to cell viability assays. n=3. (C-D) HCT116 cells were pretreated for 8 hr with 12 μM PMX205, 10 μM JPE-1375 and 2 μM Avacopan. Cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 hr and 16 hr and were subjected to apoptosis assay (C) and immunoblotting (D), respectively. n=3. ( E ) Schematic representation of experimental design for F and H–J. Created in BioRender.com. ( F ) RKO cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 16 hr, and subjected to FACS, with (right) or without (left) permeabilisation. n=3. ( G ) RKO cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 hr, and subjected to triple immunofluorescence. C5aR1 (red), Phalloidin (green), or DAPI (blue). Scale bar, 10 µm. ( H ) After treatment with 10 µg/mL tunicamycin (Tuni) or vehicle (DMSO) for 24 hr, HCT116 cells were subjected to FACS with (right) or without (left) permeabilisation. n=3. ( I ) HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) in the presence of 100 µM Dynasore and subjected to FACS with (right) or without (left) permeabilisation. n=3. ( J ) HCT116 cells were transfected with either siRNA against C5 (siC5) or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 hr, and subjected to FACS with (right) or without (left) permeabilisation. n=3. ( K ) Working model: In cancer cells, UPR-induced C5aR1 is internalised and accumulated by endocytosis under hypoxia. Intracellular C5aR1 contributes to cancer cell survival by modulating autophagy and apoptosis under hypoxia. To effectively target the C5a/C5aR1 axis in the TME, cell permeable C5aR1 inhibitors may be more effective.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 Inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 hr, or with C5aR1 antagonists, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639) for 8 hr, respectively.

Techniques: Two Tailed Test, Cell Culture, Apoptosis Assay, Western Blot, Immunofluorescence, Transfection

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: C5aR1 is mainly localized in unmyelinated fibers. Longitudinal and cross‐sections of human sural nerves were labeled for C5aR1 (red, A; green, D), Caspr (green, B), and NCAM (E, red). Double‐stained images are merged (C, F). Teased mouse sciatic nerve images of labeled C5aR1 (red, G, J) and Caspr (green, H). Double‐stained mouse sciatic fibers were merged (I). Peripherin (green, K) merged (L). Caspr‐contactin‐associated protein; NCAM‐neural cell adhesion molecule; n = 10 mice; Scale bar 10, 20 μM.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Labeling, Staining

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: mRNA and protein expression in the mouse sciatic nerve. Quantitative PCR (qPCR) analysis and western blotting were performed on sciatic nerves from 13‐week‐old mice ( n = 6 nerves). Intrinsic expression of the complement receptors and factors is seen (A). The outcomes are presented relative to hypoxanthine guanine phosphoribosyltransferase expression using the 2 −ΔCT method calculation method. Proteins C3aR and C5aR1 are present in mouse sciatic nerves ( n = 6 nerves) (B, C). HPRT‐hypoxanthine guanine phosphoribosyltransferase; ** p < 0.01.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell Death & Disease

Article Title: Intracellular C5aR1 inhibits ferroptosis in glioblastoma through METTL3-dependent m6A methylation of GPX4

doi: 10.1038/s41419-024-06963-5

Figure Lengend Snippet: A TEM was used to visualize ferroptosis in U87 and U251 cells. Scale bars, 2 μm, and 500 nm. B Protein expression of GPX4 after C5aR1 knockdown, and the related quantitative analysis, ( n = 3, means ± SD, ** P < 0.01, *** P < 0.001). C Protein expression of GPX4 after treatment with different concentrations of PMX205, and the related quantitative analysis, ( n = 3, means ± SD, * P < 0.05). D Abundance of 4-HNE after C5aR1 knockdown, and the related quantitative analysis, ( n = 3, means ± SD, ** P < 0.01). E Intracellular MDA levels after C5aR1 knockdown, and the related quantitative analysis, ( n = 3, means ± SD, * P < 0.05). F GSH level after C5aR1 knockdown, and the related quantitative analysis, ( n = 3, means ± SD, * P < 0.05). G Relative ROS levels after C5aR1 knockdown, and the related quantitative analysis, ( n = 3, means ± SD, * P < 0.05). H Oxidation-induced fluorescence of C11-BODIPY 581/591 in U87/U251 cells transfected with si-C5aR1. Green fluorescence indicates the oxidation reaction (O-BOD), while red fluorescence indicates the reduction reaction (N-BOD) The related quantitative analysis is shown, ( n = 3, means ± SD, ** P < 0.01). Scale bar, 50 μm.

Article Snippet: The apoptosis inhibitor Z-VAD-FMK (HY-16658B, MedChemExpress MCE USA), the ferroptosis inhibitors ferrostatin-1 (HY-100579, MCE, USA) and liproxstatin-1 (HY-12726, MCE, USA), the necroptosis inhibitor necrostatin-1 (HY-15760, MCE, USA), the autophagy inhibitor 3-methyladenine (HY-19312, MCE, USA) and the C5aR antagonist PMX205 (HY-110136A, MCE, USA) were used.

Techniques: Expressing, Knockdown, Fluorescence, Transfection

Journal: Medical Microbiology and Immunology

Article Title: Exploration of compounds to inhibit the Panton-Valentine leukocidin of Staphylococcus aureus

doi: 10.1007/s00430-024-00803-1

Figure Lengend Snippet: Half-maximal effective concentrations (EC 50 ) of Panton-Valentine leukocidin (PVL) at different concentrations of potential competitors

Article Snippet: These compounds were avacopan/CCX168 (C5aR antagonist, Thermo Fisher Scientific, Waltham, MA, USA), BM213 (C5aR agonist, MedChemExpress, Monmouth Junction, NJ, USA), DF2593A (C5aR antagonist, Sigma-Aldrich, St. Louis, MO, USA), JPE-1375 (C5aR antagonist, MedChemExpress), PMX205 (C5aR antagonist, Hycultec, Beutelsbach, Germany), W-54,011 (C5aR antagonist, MedChemExpress) as well as NQ301 (CD45 inhibitor, MedChemExpress).

Techniques: Concentration Assay, Control