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: Scientific Reports

Article Title: Complement component 5 promotes lethal thrombosis

doi: 10.1038/srep42714

Figure Lengend Snippet: Mice were injected with histone or saline (45 μg/g, n = 6 per group). The blood samples were collected at 1 h after injection. The profiles of CD11b-CD45 positive cells and CD41-CD45 complex (platelet/leukocyte aggregates; PLAs) are shown in figure ( A to B ) or ( D to E ), respectively. The average rates are also shown in figure ( C and F ), respectively. The concentration of C5a are shown in figure ( G ). C5a was not detected in C5 deficient mice. The figures show the experimental means ± SD. ** P < 0.01 vs Histone group of C5 deficient, †† P < 0.01 vs Vehicle group of C5 sufficient (Scheffe’s test).

Article Snippet: The mice received a single tail-vein injection of the C5a receptor antagonist PMX205 (Tocris Bioscience, Bristol) (50 μg/body) or saline.

Techniques: Injection, Saline, Concentration Assay

Journal: Scientific Reports

Article Title: Complement component 5 promotes lethal thrombosis

doi: 10.1038/srep42714

Figure Lengend Snippet: The mice were received a single tail-vein injection of C5a receptor antagonist PMX205 (50 μg/body) or saline. At 10 min after injection of saline or PMX205, the mice in the histone and PMX 205 groups were injected with unfractionated histones. The mice in the control group were injected with saline only. Liver function were defined as AST, ALT and LDH levels, and these are shown in ( A – C ), respectively. The profiles of CD41-CD45 complex (platelet/leukocyte aggregates; PLAs) and CD11b-CD45 positive cells are shown in figure ( D , E ), respectively. The figures show the experimental means ± SD. * P < 0.05 vs PMX205 group (Scheffe’s test).

Article Snippet: The mice received a single tail-vein injection of the C5a receptor antagonist PMX205 (Tocris Bioscience, Bristol) (50 μg/body) or saline.

Techniques: Injection, Saline, Control

Journal: Scientific Reports

Article Title: Complement component 5 promotes lethal thrombosis

doi: 10.1038/srep42714

Figure Lengend Snippet: The black dotted arrow show previously known interactions of these systems. The black solid arrows identify the new paths in the present study. Histones induce platelet aggregation and coagulation disorder; the coagulation disorder promotes cleavage of C5. C5a increases the expression of CD11b (CR3). CR3 binds to CD41 (GPIIb), and this aggregations induce embolism. Embolism accelerates liver injury.

Article Snippet: The mice received a single tail-vein injection of the C5a receptor antagonist PMX205 (Tocris Bioscience, Bristol) (50 μg/body) or saline.

Techniques: Coagulation, Expressing