Journal: bioRxiv

Article Title: MIIP downregulation promotes colorectal cancer progression via inducing adjacent adipocytes browning

doi: 10.1101/2023.01.28.526013

Figure Lengend Snippet: A. The concentration of AZGP1 in the supernatant of HCT116 cells with different MIIP expression levels was determined by ELISA (7 biological replicates per group). B. The concentration of Azgp1 in the supernatant of stable Miip knockdown CMT93 or CT26.WT cells was determined by ELISA (7 biological replicates per group). C. The mRNA levels of AZGP1/Azgp1 in cells with different MIIP/Miip expression levels was determined by RT-qPCR (3 biological replicates per group). D. Co-immunoprecipitation analysis of MIIP and AZGP1 in HCT116 cells transfected with pCMV-MIIP-FLAG, by immunoprecipitation with anti-FLAG and immunoblot with anti-AZGP1. E. Immunoblots of phospho-HSL and UCP1 in differentiated mature adipocytes treatment with the CM from AZGP1 -knockdown or NC HCT116-MIIP +/- cells for 24 h (Representative of 3 biological replicates per group). F. Mature adipocytes were treated with the CM from AZGP1 -knockdown or NC HCT116-MIIP +/- cells for 24 h, then washed and medium was changed. After additional 24 h, concentrations of free fatty acids (Left) and glycerol (Right) in supernatants were detected. G. HCT116 cells were co-transfected with pEX-3-AZGP1-6×His and pCMV-MIIP-FLAG (or empty vector) for 48 h, then ER and Golgi apparatus protein were extracted, followed by co-immunoprecipitation analysis. The lysates were precipitated with anti-His and immunoblot with anti-AZGP1 and anti-MIIP. H. HCT116 cells were transfected with the ectopic expression plasmid of AZGP1 WT and its NQ mutants for 48 h, followed by co-immunoprecipitation analysis with anti-MIIP and anti-His successively. I. Glycosylation pattern of AZGP1 protein in HCT116 control and MIIP-overexpressing cells. Cell lysates were treated with PNGase F and analyzed by immunoblot analysis (Representative of 3 biological replicates per group). J. Glycosylation of AZGP1 protein in HCT116 control and MIIP-overexpressing cells. Cells were treated with Tunicamycin (1 μg/mL) or not and subjected to immunoblot analysis (Representative of 3 biological replicates per group). Data information: All data are shown as mean ± SD (A-C, F). Two-tailed unpaired Student’s t-test. *** P < 0.001.

Article Snippet: ER-Tracker Green (C1042S), Anti-FLAG Magnetic Beads (P2115), Anti-His Magnetic Beads (P2135) and Mouse IgG Magnetic Beads (P2171) were purchased from Beyotime Biotechnology (Shanghai, China).

Techniques: Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, Two Tailed Test