Fig. 1: Identifying the deubiquitinase USP2 as a positive regulator for CD47.

a Overview of the outline for establishing H1975 and HEK293T cell lines expressing a fusion GFP gene in the C-terminus of CD47 (CD47-GFP) and subsequently identifying the deubiquitinase of CD47 through DUB siRNA library screening with two stable cell lines. DUBs deubiquitinases; FACS fluorescence-activated cell sorting; MFI mean fluorescence intensity. b Whole-cell lysates (WCL) of H1975 or PC9 cells treated with indicated deubiquitinase inhibitors (2.5 µM) or dimethyl sulfoxide (DMSO) for 12 hours (h) were prepared and subjected to immunoblotting (IB) analysis. c–e IB analysis of WCL derived from H1975 cells (c) and PC9 cells (e) treated with ML364 (1 µM and 2 µM) or DMSO for 16 h. The mRNA level of CD47 in H1975 cells (c) was measured using reverse transcription quantitative PCR (RT-qPCR) (d). f, g Immunofluorescence (IF) staining for CD47 in H1975 cells (f) and PC9 cells (g) treated with ML364 (2 µM) for 16 h. Scale bar, 25 μm. h–k IB analysis of WCL derived from H1975 (h) or PC9 (j) cells stably expressing shUSP2 or shGFP, respectively. The mRNA level of CD47 in H1975 (i) or PC9 (k) cells was measured using RT-qPCR. l, m IB analysis of Cd47 protein expression in the tissues of lung, heart, or kidney obtained from wild-type (WT) and Usp2^−/− mice (l). Quantification of Cd47 protein band intensity was normalized to vinculin (m). n–p IB analysis of WCL derived from HEK293T cells co-transfected with indicated constructs (n, p). The mRNA level of CD47 was quantified by RT-qPCR (o). EV: empty vector. q, r IB analysis of WCL derived from HEK293T cells co-transfected with indicated constructs. Cells were treated with 200 μg/ml CHX for the indicated time points (q). Quantification of CD47 protein band intensity was normalized to vinculin, then compared to the t = 0 time point (r). s, t Representative images from immunohistochemical (IHC) staining of CD47 and USP2 in human lung adenocarcinoma (s). Scale bar, left panels: 100 μm; right panels: 50 μm. n = 83. Quantification of USP2 and CD47 staining intensities was performed as average optical density (AOD) [AOD = Integrated Optical Density (IOD) SUM/Area SUM] (t). For (d, i, k, m, o, and r), unpaired two-tailed Student’s t-test. Correlations were analyzed by Pearson’s test (t). Data are shown as the mean ± SD, n = 3 independent biological replicates. P < 0.05 was considered statistically significant. n = 3 biologically independent experiments for (b, c, e, h, j, l, n, p, and q). Source data are provided as the Source Data file.