Fig. 6: IRE1α suppresses Thrombospondin 1 expression to promote endothelial cell proliferation.

a–f MS1 endothelial cells (a, c, e) or primary HUVECs (b, d, f) were infected with lentiviruses encoding a shRNA directed against IRE1α (shErn1 or shERN1) or a scramble control (shCtrl) for 48 hours. a, b Quantitative RT-PCR analysis of Xbp1 mRNA splicing and Thbs1/THBS1 mRNA abundance in MS1 cells (a) or HUVECs (b) pre-cultured for 4 hours at 5 mM glucose or following stimulation for 24 hours with high glucose at 16 mM (n = 3 independent experiments). c, d Immunoblot analysis of IRE1α and TSP1 protein levels in MS1 cell lysates (c) or HUVEC lysates (d) when cultured at 5 mM versus 16 mM glucose for 24 hours. α-Tubulin was used as a loading control (n = 3 independent experiments). e, f Immunoblot analysis of TSP1 protein in culture medium of MS1 cells (e) or HUVECs (f). Cell lysate α-Tubulin was used as a loading control (n = 3 independent experiments). g Cell proliferation and viability analysis by cell-count assay or using the CCK-8 Kit for lentivirus-infected MS1 cells cultured at 16 mM glucose (n = 2 independent experiments). h, i MS1 cells were infected with shCtrl, shErn1, or both shErn1 and shThbs1 lentiviruses for 48 hours. h Immunoblot analysis of IRE1α and TSP1 proteins in MS1 cell lysates. i MS1 cell proliferation analysis by cell-count assay or using the CCK-8 Kit when cultured at 16 mM glucose (n = 2 independent experiments). j Proliferation analysis by the CCK-8 Kit of MS1 cells that were cultured at 16 mM glucose and treated with PBS (Vehicle, Veh.) or 1 μg/mL recombinant human TSP1 protein in the absence or presence of 1 μg/mL anti-CD47 neutralizing antibody for 48 hours (n = 6 independent treatment experiments). Data are shown as mean ± SEM by two-way ANOVA (a–d, g, i, * indicates shCtrl versus shErn1; # indicates shErn1 versus shErn1/shThbs1 in i) or unpaired two-tailed Student’s t-test (e, f, j).