Fig 1: Caspase-1 activation enhances glycolysis and impairs cell differentiation. (A) Schematic model of PMA induced differentiation of THP-1 monocytes into macrophages. This figure was generated using pictures provided by the Servier Medical Art homepage https://smart.servier.com/. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. (B) QPCR analyses were performed to quantify mRNA expression level of C1qbp, Fis1, Slc2a1, Ldha, and Ki67. (C) Western blot analysis of whole protein extracts from PMA-induced differentiated THP-1 macrophages using gC1qR-directed antibodies specific for epitopes located in exon 1, 3, or 6. (D) Secretion of IL-1β (left panel), gC1qR (middle panel) or lactate (right panel) was determined using specific ELISA using supernatants from untreated or LPS stimulated PMA-differentiated THP-1 cells in the presence or absence of caspase-1 inhibitor (10 μg/ml; Ac-YVAD-cmk from InvivoGen). (E) GC1qR protein expression in mitochondrial/ cell membrane protein fractions was quantified by Western blot experiments. PMA-differentiated THP-1 cells were stimulated with LPS in the presence or absence of caspase-1 inhibitor (10 μg/ml; Ac-YVAD-cmk) or were left untreated. Densitometry was performed using the software ImageJ (right panel). (F) Schematic model of butyrate induced goblet cell differentiation of HT29-MTX cells (left panel). Oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) of HT29-MTX cells stimulated with 1.25 mM butyrate for 24 h or left untreated were determined using the Seahorse XF Cell Mito Stress Test (right panel). (G) HT29-MTX cells were incubated in the absence or presence of 1.25 mM butyrate for 72 h. After incubation, cells were counted. (H) HT29-MTX cells were transiently transfected for 96 h with plasmids encoding full-length human caspase-1 (C), human ASC (A), human NLRP3 (N) or with a mock plasmid in the presence or absence of 1.25 mM butyrate. Whole protein extracts were separated by SDS-PAGE under reducing conditions and Western blot experiments were performed using indicated primary antibodies. Results are presented as mean ± SEM from at least three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Fig 2: The effect of overexpression of gC1qR on mitochondrial function of human colonic NCM 460 epithelial cell lines. The human colonic epithelial cells were transfected with gC1qR vector or empty vector for 48 h. a Intracellular ROS generation was determined by fluorescence of H2DCFDA (Green). b The change of mitochondrial membrane potential was detected. The relative Δψm value was measured by monitoring the fluorescence of JC-1 (590: 527 nm fluorescence ratio). c ATP content was detected in NCM 460 epithelial cell. The data are means ± S.D. of three separate experiments performed in triplicate. **p < 0.01, #p > 0.05 versus Mock group
Fig 3: Immunohistochemical staining of HABP1 in breast tissuesHABP1 immunoreactivity was observed mainly in the cytoplasm. Staining for each specimen is shown at two magnification: top, 100×; bottom, 200×. HABP1 protein low expression specimens (A, C); HABP1 protein low expression specimens (B, D).
Fig 4: GC1qR expression is upregulated in CRC tissues and negatively correlates with the tumors' differentiation state. (A–C) QPCR analyses were performed to quantify mRNA expression level of (A) Caspase-1, (B) C1qbp, or (C) Ki67 in 10 paired colonic normal or tumor tissues collected from CRC patients. (D) Results received from qPCR experiments for Caspase-1 mRNA expression were put into relation with Ki67 mRNA expression level. (E,F) C1qbp mRNA expression was quantified by qPCR experiments in a set of CRC tumor samples of different tumor (E) stage and (F) grade. (G) Immunohistochemistry analyses of tumor tissues displaying different grading states [grade 1 (G1), grade 2 (G2), grade 3 (G3), grade 4 (G4)] collected from CRC patients using primary antibodies specific for gC1qR (clone EPR8871; 63x magnification), gC1qR exon 6 (63x magnification), TOM22 (63x magnification) or Caspase-1 (20x magnification). **p ≤ 0.01.
Fig 5: GC1qR displays two caspase-1 cleavage sites. (A) Schematic structure model of the human gC1qR protein. SP, signal peptide sequence for mitochondrial import. Homology model of human gC1qR protein was generated using the PHYRE2 server. Bright green = predicted mitochondrial leader sequence, dark green = residual amino acid (aa) residues of exon 1, gray = mature gC1qR. (B) Crystal structure of trimeric human gC1qR was generated based on pdb file pdb1p32. (C) Immunohistochemistry analyses of paraffin-embedded formalin-fixed HT29 cells using anti-gC1qR Ab clone 60.11 or anti-gC1qR Ab clone EPR8871. (D) Co-localization of gC1qR with mitochondrial HSP60 protein in HAP1 cells was assessed by fluorescence microscopy using the anti-gC1qR clone EPR8771. (E) In silico prediction of potential protease cleavage sites was performed using the PeptideCutter software (https://web.expasy.org/peptide_cutter/). Highlighted in pink = predicted caspase-1 cleavage site at amino acid D77; highlighted in yellow = predicted caspase-1 cleavage site at amino acid D229. (F) Predicted caspase-cleavage sites at D77 and D229 were highlighted in pink or yellow, respectively, in the generated homology model of gC1qR. (G) Representative pictures from immunohistochemistry analyses of five independent paraffin-embedded formalin-fixed human colonic biopsy samples collected from normal tissue sites from CRC patients using anti-gC1qR Ab (clone EPR8871), anti-TOM22 Ab or anti-Caspase-1 Ab.
Supplier Page from Abcam for Anti-GC1q R antibody [EPR8871]