Journal: bioRxiv

Article Title: Consumption of processed foods impairs memory function through dietary advanced glycation end-products

doi: 10.64898/2026.01.07.698065

Figure Lengend Snippet: a , Illustration of the electrophysiology field potential recording setup. b , Ratio of field to volley from electrophysiology field potential recordings of dorsal hippocampus (HPCd) brain tissue slices in rats after 30-d early life AGE-rich diet (n=14 CTL tissue slices, n=15 AGE-rich tissue slices; 2-way repeated measures ANOVA with factors of diet group, fiber volley amplitude [repeated measure], and diet group × fiber volley interaction; P=0.04 diet group, P<0.0001 fiber volley amplitude, P=0.99 diet group × fiber volley amplitude). c , mRNA expression of complement component 3 (C3) in the HPCd (n=7 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.04). d , mRNA expression of complement component 5 (C5) in the HPCd (n=7 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.68). e , mRNA expression of C3aR in the HPCd (n=7 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.17). f , mRNA expression of C5aR1 in the HPCd (n=8 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.16). g , Structures of the peptide required for complement system receptor activation and of AGEs elevated in the heat-treated diet. h , C5aR1 pERK 1/2 signaling in response to MG-H1, CML, and CEL (smoothed lines indicate nonlinear least squares fit for each treatment). i , C5aR1 Receptor-G protein-Bioluminescence Resonance Energy Transfer (RG-BRET) assay for binding affinities between C5a and MG-H1, CML, CEL, or the C5aR1-selective antagonist avacopan (smoothed lines indicate nonlinear least squares fit for each treatment). Error bars represent standard error of the mean (SEM). *P<0.05. All n’s indicate number of rats or slices per group. Additional details about the statistical analyses for each subpanel can be found in . AGE, advanced glycation end-product; ANOVA, analysis of variance; C3, complement component 3; C3aR, receptor for complement component 3a; C5, complement component 5; C5aR1, receptor for complement component 5a; CA1, Cornu Ammonis subfield 1; CA3, Cornu Ammonis subfield 3; EC, entorhinal cortex; CEL, carboxyethyllysine; CML, carboxymethyllysine; CTL, control [diet]; HPCd, dorsal hippocampus; MG-H1, methylglyoxal-derived hydroimidazolone; pERK, phospho-extracellular signal-regulated kinase; PN, postnatal day; RG-BRET, Receptor-G protein-Bioluminescence Resonance Energy Transfer.

Article Snippet: The following Applied Biosystems probes for use with rats were used: C3 (Rn00566466_m1), C5 (Rn01436156_m1), C3aR (Rn00583199_m1), C5aR1 (Rn02134203_s1), RAGE (Rn01525753_g1), NfĸB (Rn01502266_m1), PSD-95 (Rn00571479_m1), occludin (Rn00580064_m1), claudin-1 (Rn00581740_m1), claudin-5 (Rn01753146_s1), and mucin 2 (Rn01498206_m1). qPCR was performed using Applied Biosystems TaqMan Fast Advanced Master Mix (Cat. no. 444557, ThermoFisher Scientific) and an Applied Biosystems QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific).

Techniques: Expressing, Two Tailed Test, Activation Assay, Bioluminescence Resonance Energy Transfer, Binding Assay, Control, Derivative Assay

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: Upregulation of C5a/C5aR1 promotes cytokine release and drives inflammation in LN mice. (A) Schematic diagram of the animal model induction process; (B) Mouse cytokine antibody array Panel (A) showing increased C5a expression; (C) Western blot analysis of C5aR1 expression in mouse renal tissues, with β‐actin as the internal control ( n = 5); (D) Immunohistochemical analysis of C5aR1 expression in paraffin‐embedded kidney sections ( n = 5); (E) Analysis of IL‐1β and TNF‐α mRNA expression in mouse kidney tissues, with β‐actin as the internal control ( n = 5). (F) Representative H&E staining of paraffin‐embedded kidney sections ( n = 5); black arrows indicate inflammatory cell infiltration around renal tubules. Group comparisons were performed using a two‐tailed unpaired t ‐test, followed by LSD or Tukey post hoc tests to determine intergroup differences. Immunohistochemistry scores were analyzed using nonparametric tests. p > 0.05, not significant. **** p < 0.0001. H&E, hematoxylin and eosin; LN, lupus nephritis; LSD, least significant difference.

Article Snippet: The LN + C5aRA1 group received PMX53, a C5aR1 antagonist (10 mg/kg; HY‐106178, MedChemExpress), once daily by oral gavage.

Techniques: Animal Model, Ab Array, Expressing, Western Blot, Control, Immunohistochemical staining, Staining, Two Tailed Test, Immunohistochemistry

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: C5aR1A alleviates inflammation and renal dysfunction in LN mice. (A) Schematic diagram of the animal model procedure; (B) mRNA expression levels of IL‐1β and TNF‐α in mouse renal tissue, normalized to β‐actin ( n = 5); (C) PAS and H&E staining of paraffin‐embedded kidney sections ( n = 5); (D, E) Immunohistochemical analysis of IL‐1β, MCP‐1, TNF‐α, and TGF‐β expression in renal tissues at 40× magnification ( n = 5); (F) Western blot analysis of PTEN and p‐AKT in mouse kidney tissue, with GAPDH as a loading control ( n = 5); (G) Quantification of BUN and Scr levels in mouse kidney tissues ( n = 5). Statistical analysis was performed using one‐way ANOVA. p > 0.05, not significant. * p < 0.05; *** p < 0.001; **** p < 0.0001. ANOVA, analysis of variance; BUN, blood urea nitrogen; H&E, hematoxylin and eosin; LN, lupus nephritis; MCP‐1, monocyte chemoattractant protein‐1; PAS, periodic acid–Schiff; PTEN, phosphatase and tensin homolog; Scr, serum creatinine; TGF‐β, transforming growth factor‐β.

Article Snippet: The LN + C5aRA1 group received PMX53, a C5aR1 antagonist (10 mg/kg; HY‐106178, MedChemExpress), once daily by oral gavage.

Techniques: Animal Model, Expressing, Staining, Immunohistochemical staining, Western Blot, Control

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: C5a suppresses PTEN expression and enhances AKT pathway activation to promote inflammation. (A) Schematic diagram of the animal model procedure; (B) Schematic diagram of the in vitro cell experiment; (C) Protein‐protein interaction network illustrating key molecules linking C5a/C5aR11 with PTEN and the PI3K/AKT signaling pathway (confidence score = 0.15); (D) Western blot analysis of C5aR1 knockdown by three siRNAs in vitro (D1–D2) and by three shRNAs in kidney tissues in vivo (D3–D4), with GAPDH as loading control ( n = 3); (E) Western blot analysis of C5aR1 and PTEN expression after C5aR1 knockdown by siRNAs in vitro (E1–E3) and by shRNAs in kidney tissues in vivo (E4–E6), with GAPDH as loading control ( n = 3). (F–H) Results of the human/mouse AKT pathway phosphorylation antibody array C1 (RayBiotech) showing the expression of BAD, PRAS40, and PTEN. Group comparisons were performed using a two‐tailed unpaired t ‐test or one‐way ANOVA. p > 0.05, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ANOVA, analysis of variance; BAD, Bcl‐2‐associated death promoter; PTEN, phosphatase and tensin homolog.

Article Snippet: The LN + C5aRA1 group received PMX53, a C5aR1 antagonist (10 mg/kg; HY‐106178, MedChemExpress), once daily by oral gavage.

Techniques: Expressing, Activation Assay, Animal Model, In Vitro, Western Blot, Knockdown, In Vivo, Control, Phospho-proteomics, Ab Array, Two Tailed Test

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: Upregulation of C5a/C5aR1 promotes cytokine release and drives inflammation in LN mice. (A) Schematic diagram of the animal model induction process; (B) Mouse cytokine antibody array Panel (A) showing increased C5a expression; (C) Western blot analysis of C5aR1 expression in mouse renal tissues, with β‐actin as the internal control ( n = 5); (D) Immunohistochemical analysis of C5aR1 expression in paraffin‐embedded kidney sections ( n = 5); (E) Analysis of IL‐1β and TNF‐α mRNA expression in mouse kidney tissues, with β‐actin as the internal control ( n = 5). (F) Representative H&E staining of paraffin‐embedded kidney sections ( n = 5); black arrows indicate inflammatory cell infiltration around renal tubules. Group comparisons were performed using a two‐tailed unpaired t ‐test, followed by LSD or Tukey post hoc tests to determine intergroup differences. Immunohistochemistry scores were analyzed using nonparametric tests. p > 0.05, not significant. **** p < 0.0001. H&E, hematoxylin and eosin; LN, lupus nephritis; LSD, least significant difference.

Article Snippet: Sections were incubated overnight at 4°C with rabbit primary antibodies, including C5aR1 (Proteintech), IL‐1β (Proteintech, Cat#16806‐1‐AP), monocyte chemoattractant protein‐1 (MCP‐1) (Abcam, Cat#ab25124), TNF‐α (Abcam, Cat#ab6671), and transforming growth factor‐β (TGF‐β) (Proteintech, Cat#21898‐1‐AP).

Techniques: Animal Model, Ab Array, Expressing, Western Blot, Control, Immunohistochemical staining, Staining, Two Tailed Test, Immunohistochemistry

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: C5a suppresses PTEN expression and enhances AKT pathway activation to promote inflammation. (A) Schematic diagram of the animal model procedure; (B) Schematic diagram of the in vitro cell experiment; (C) Protein‐protein interaction network illustrating key molecules linking C5a/C5aR11 with PTEN and the PI3K/AKT signaling pathway (confidence score = 0.15); (D) Western blot analysis of C5aR1 knockdown by three siRNAs in vitro (D1–D2) and by three shRNAs in kidney tissues in vivo (D3–D4), with GAPDH as loading control ( n = 3); (E) Western blot analysis of C5aR1 and PTEN expression after C5aR1 knockdown by siRNAs in vitro (E1–E3) and by shRNAs in kidney tissues in vivo (E4–E6), with GAPDH as loading control ( n = 3). (F–H) Results of the human/mouse AKT pathway phosphorylation antibody array C1 (RayBiotech) showing the expression of BAD, PRAS40, and PTEN. Group comparisons were performed using a two‐tailed unpaired t ‐test or one‐way ANOVA. p > 0.05, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ANOVA, analysis of variance; BAD, Bcl‐2‐associated death promoter; PTEN, phosphatase and tensin homolog.

Article Snippet: Sections were incubated overnight at 4°C with rabbit primary antibodies, including C5aR1 (Proteintech), IL‐1β (Proteintech, Cat#16806‐1‐AP), monocyte chemoattractant protein‐1 (MCP‐1) (Abcam, Cat#ab25124), TNF‐α (Abcam, Cat#ab6671), and transforming growth factor‐β (TGF‐β) (Proteintech, Cat#21898‐1‐AP).

Techniques: Expressing, Activation Assay, Animal Model, In Vitro, Western Blot, Knockdown, In Vivo, Control, Phospho-proteomics, Ab Array, Two Tailed Test

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

Figure Lengend Snippet: A Representative images of C5aR1 staining in TMAs for prostate, endometrial, and colorectal normal and tumour tissues. A zoomed in region of the TMA is shown to the right. Scale bar, 100 µm. B H-score of staining for TMAs in ( A ) is shown. Individual dots show data for different cores analysed for each patient. 3 prostate, 4 endometrial and 5 colorectal cancer patient samples were analysed. C Pearson’s correlation of C5aR1 mRNA expression and Ahmed Hypoxia signature in TCGA colorectal cancer and glioblastoma samples. R score and p -value are shown. D , E HCT116 ( D ) and LN229 ( E ) cells were cultured under the indicated oxygen conditions for 24 h and 16 h, respectively, and subjected to qRT-PCR. n = 3. Individual biological replicates (large points) represent the average of the technical replicates (small points). p -values were calculated using biological replicates by one-way ANOVA with Dunnett’s test. F , G Serial sections of HCT116 spheroids treated with EF5 were stained with the indicated antibodies; C5aR1 (red), a hypoxia marker, EF5 (green), or DAPI (blue). The dotted line represents the estimated outside edge of the EF5-positive regions. Scale bar, 50 µm.

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 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Staining, Expressing, Cell Culture, Quantitative RT-PCR, Marker

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

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 by two-tailed paired Student’s t -test ( A ), two-tailed unpaired Student’s t -test ( B − D ), two-way ANOVA with uncorrected Fisher’s LSD test ( E – G and I ), or one-way ANOVA with Dunnett’s test ( H ). A HIF-1α- KO and WT HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h and subjected to qRT-PCR. n = 3 and 4. B – D HCT116 cells were treated with either 200 µM cobalt chloride (CoCl 2 ) for 24 h ( B ), 10 µg/mL tunicamycin (Tuni) for 24 h ( C ), 2 µM thapsigargin (Thap) for 16 h ( D ), or its vehicle (Ctrl), and subjected to qRT-PCR. n = 3. E – G HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) in the presence of either IRE1-inhibitor (E), PERK-inhibitor ( F ), or ATF6-inhibitor ( G ), and subjected to qRT-PCR. n = 3. H HCT116 cells were transfected with the indicated siRNA or scramble siRNA (siScr), cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to qRT-PCR. n = 4. I After simultaneously silencing XBP1, ATF4, and ATF6 using siRNA mixtures, HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to qRT-PCR. n = 3. J Enrichment Analysis with public ChIP-seq data on ChIP-Atlas website is shown ( https://chip-atlas.org/peak_browser ). Colours represent copy numbers of the indicated transcription factors binding to the C5aR1 gene locus in all cell lines. K Pearson’s correlation of C5aR1 mRNA expression and Xhu UPR signature in TCGA colorectal cancer and glioblastoma samples. R score and p -value are shown. L , M Serial sections of HCT116 spheroids treated with EF5 ( L ) or HCT116 tumour xenografts ( M ) were stained with the indicated antibodies; ( L ) Section 1, C5aR1(red), BiP (green), or DAPI (blue); Section 2, BiP (red), EF5 (green), or DAPI (blue). (M) C5aR1 (red), BiP (green), or DAPI (blue). Scale bar, 50 µm.

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 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Cell Culture, Quantitative RT-PCR, Transfection, ChIP-sequencing, Binding Assay, Expressing, Staining

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

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 by two-way ANOVA with uncorrected Fisher’s LSD test ( C , D , and F ) or two-tailed paired Student’s t -test ( G – J ). A , B HCT116 cells were transfected with either siC5aR1 or siScr ( A ) or with either pcDNA3.1/C5aR1-GFP (C5aR1) or its empty vector (EV) ( B ), cultured under normoxia or hypoxia (<0.1% O 2 ) for the indicated periods, and subjected to immunoblotting. C RKO cells were transfected with siC5aR1 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to immunocytochemistry and apoptosis assay. C5aR1 (red), Phalloidin (green), or DAPI (blue). Scale bar, 10 µm. n = 3. D HCT116 cells were transfected with either siC5aR1 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h and subjected to apoptosis assay. n = 3. E HCT116 cells were transfected with either siRNA against C5 (siC5) or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for the indicated periods, and subjected to immunoblotting. F RKO cells were transfected with either siC5 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to apoptosis assay. n = 3. G , H HCT116 cells were transfected with either siC5aR1 or siScr ( G ) or with either pcDNA3.1/C5aR1-GFP (C5aR1) or EV ( H ), cultured under normoxia or hypoxia (<0.1% O 2 ) for 40 h ( G ) and 32 h ( H ), respectively, and subjected to cell viability assay. n = 3. I , J HCT116 ( I ) and RKO ( J ) cells were transfected with either siC5aR1 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h and subjected to clonogenic survival assay. n = 3.

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 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Immunocytochemistry, Apoptosis Assay, Viability Assay, Clonogenic Cell Survival Assay

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

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 by one-way ANOVA with Dunnett’s test ( A , B , and E ; DMSO vs JPE-1375 and Avacopan), two-way ANOVA with uncorrected Fisher’s LSD test ( C , H ), and two-tailed paired Student’s t -test ( E ; Water vs PMX205), two-way ANOVA with Tukey test ( F ). A , B HCT116 cells were pretreated for 8 h with the indicated dose of C5aR1 antagonists: PMX205, JPE-1375 or Avacopan. Cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 40 h and subjected to cell viability assays. n = 3. C – E HCT116 cells were pretreated for 8 h with 12 μM PMX205, 10 μM JPE-1375 or 2 μM Avacopan. Cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h ( C and E ) and 16 h ( D ), and were subjected to apoptosis assay ( C ), immunoblotting ( D ), and clonogenic survival assay ( E ), respectively. n = 3. F Diameters of HCT116 spheroids were measured daily after the administration of the indicated drug. Representative bright-field microscopy images (left) and changes in diameter (right) of spheroids are shown. n = 12 ( G , H ) HCT116 spheroids were treated with vehicle or Avacopan and EF5. Sections were subjected to TUNEL assay and immunohistochemistry. Apoptosis (red), EF5 (green), or DAPI (blue). Scale bar, 50 µm. Representative images ( G ) and red fluorescence score in EF5 positive and negative regions ( H ) are shown. n = 6–7.

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 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Cell Culture, Apoptosis Assay, Western Blot, Clonogenic Cell Survival Assay, Microscopy, TUNEL Assay, Immunohistochemistry, Fluorescence

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

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 by two-way ANOVA with uncorrected Fisher’s LSD test ( E and F ), and two-tailed paired Student’s t -test ( B and D ). A Schematic representation of experimental design for B and D–F. Created in BioRender.com. B RKO cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 16 h, and subjected to FACS, with (right) or without (left) permeabilisation. n = 3. C RKO cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to immunocytochemistry. C5aR1 (red), Phalloidin (green), or DAPI (blue). Scale bar, 10 µm. D After treatment with 10 µg/mL tunicamycin (Tuni) or vehicle (DMSO) for 24 h, HCT116 cells were subjected to FACS with (right) or without (left) permeabilisation. n = 3. E 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. F HCT116 cells were transfected with either siRNA against C5 (siC5) or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to FACS with (right) or without (left) permeabilisation. n = 3. G Working model: In hypoxic cancer cells, UPR-induced C5aR1 is internalised and accumulates by endocytosis. 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 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Cell Culture, Immunocytochemistry, Transfection