Fig 1: Annexin A1 production is enhanced during zymosan-induced inflammation. Mice were injected i.p. with zymosan (Zym) and peritoneal lavage supernatant, peritoneal macrophages (PM), and spleens were collected at 6, 24, or 72 h after Zym injection. (A,B) The levels of Annexin A1 (AnxA1) and TNF-α-stimulated gene-6 (TSG6) mRNA over time in PM and spleens analyzed by real-time PCR and normalized to that of hypoxanthine guanine phosphoribosyl transferase (Hprt) mRNA. (C,D) The abundance of AnxA1 and TSG6 in peritoneal lavage fluid (PLF) as assessed by ELISA. (E) Immunoblots analysis of AnxA1 in spleen homogenates. Lower pannel: Densitometric analysis of AnxA1 normalized to that of β-actin. Values represent the means ± SEM of results from five (A,B) or three mice (C–E). * p < 0.05, *** p < 0.001 compared with control at a given time point (Student’s t-test).
Fig 2: Annexin A1 enhances STAT6 activation and PPARγ expression and activation in BMDM and peritoneal macrophages. Immunoblots analysis of the relative amounts of phosphorylated STAT6/total STAT6 in mouse BMDM (A) and peritoneal macrophages (F) stimulated with Annexin A1 (AnxA1) for 2 h. Densitometric analysis of the relative abundance of the indicated proteins normalized to that of STAT6. Left: Immunofluorescence staining of phosphorylated STAT6 (green) in BMDM (B) and peritoneal macrophages (G) after 100 ng/mL AnxA1 treatment. The imaging medium was Vectashield fluorescence mounting medium containing DAPI. Original magnification: ×400. Scale bars = 40 μm. Representative results from three independent experiments are shown. Right: The ratio of phospho-STAT6 (+) over DAPI (+) macrophages is presented in the bar graph. Immunoblots analysis of the relative amounts of PPARγ, CD36, and β-actin in BMDM (C,E) and peritoneal macrophages (H,J) stimulated with AnxA1 for 24 h. Densitometric analysis of the relative abundance of the indicated proteins normalized to that of β-actin. PPARγ activity in nuclear extracts from BMDM (D) and peritoneal macrophages (I) was analyzed at 24 h after AnxA1 treatment as described in the Methods. Values represent the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control (Student’s t-test).
Fig 3: TRNP1 knockdown enhances the therapeutic response of HCC to PD-1 blockade. (A) The correlation between the gene expression of TRNP1 and PDCD1 in human HCC samples using the GEPIA web tool. (B) Western blot images (left) and quantitative analysis (right) of TRNP1 knockdown in Hepa 1-6 cells are shown below. The representative images (C), tumor weight (D) and tumor growth (E) of xenograft tumors from Hepa 1-6 cells with NC and shTRNP1 with/without anti-PD-1. (F) Lollipop graph illustrates the correlation between immune infiltration and TRNP1 expression. (G) The proportion of M1 and M2 macrophages in NC and TRNP1 knockdown Hepa 1–6 subcutaneous tumors was detected by flow cytometry, along with statistical data. (H) Volcano plot of ANXA1 expression in Sk-hep1 cells with or without TRNP1 knockdown. q-PCR (I) and ELISA (J) analyses show the expression of ANXA1 in mouse subcutaneous tumor tissues. ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ANXA1, annexin A1; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GEPIA, Gene Expression Profiling Interactive Analysis; HCC, hepatocellular carcinoma; NC, negative control; PD-1, programmed cell death protein 1; q-PCR, quantitative real-time polymerase chain reaction; TRNP1, TMF1-regulated nuclear protein 1.
Supplier Page from Abcam for Mouse Annexin A1 ELISA Kit