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. 2022 Mar 31;298(5):101888. doi: 10.1016/j.jbc.2022.101888

Figure 4.

Figure 4

Pepstatin A penetratin increases A2AR expression in mouse macrophages. IPMΦ cells were treated with Pepstatin A penetratin (1, 3, and 9 μM) in the absence or presence of LPS (100 ng/ml) for 4 h. A, immunofluorescence detection of A2AR in IPMΦ cells. A2AR was stained using a specific primary antibody, and Alexa-488–conjugated secondary antibody demonstrates the localization of endogenous A2AR protein (green). Nuclei of macrophages were stained with DAPI (blue). Images were acquired using the Opera Phenix High Content Confocal System (PerkinElmer); 190 fields and 2000 to 3000 cells were acquired per well, and laser-based autofocus was performed for each imaging position. Images of DAPI and Alexa-488 channels were collected at 2 μm of Z image plane using a 63× water immersion objective (NA, 1.15) to visualize the cells and the localization of A2AR. B, the primary data were analyzed using Harmony 4.8 software (PerkinElmer). Spot Analyses Ready to Made Solution (http://www.perkinelmer.com/product/harmony-4-2-office-hh17000001) was used with custom modifications. Image intensities were rescaled, and cells were identified using the DAPI signal. Cellular phenotypes were characterized based on the Alexa-488 signal. Cellular features, such as the number of spots, relative spot intensity in the membrane, and cytoplasm regions, were extracted. Statistical analyses of parallel datasets were made using GraphPad Prism 8 program. The evaluation of the data based on the individual analyses of 2000 to 3000 different cells is presented as mean ± SD. Values from ANOVA: F = 20.53 and p < 0.001, F = 24.78 and p < 0.001 for number of spots in membrane, cytoplasm region, respectively; F = 9.52 and p < 0.001, F = 35.28 and p < 0.001 for relative spot intensity in membrane, cytoplasm region, respectively. ∗∗p < 0.01 and ∗∗∗p < 0.001 versus control (vehicle treated); ##p < 0.01, ###p < 0.001 versus LPS-treated cells. A2AR, adenosine A2A receptor; DAPI, 4′,6-diamidino-2-phenylindole; IPMΦ, mouse peritoneal macrophage; LPS, lipopolysaccharide; NA, numerical aperture.