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. 2022 Jun 7;25(7):104547. doi: 10.1016/j.isci.2022.104547

Figure 6.

Figure 6

FactorXa/PAR1 signaling promotes the dysfunction of brown adipocytes

(A-H) Nivo Multimode Microplate Reader study analyzing signal of mitochondrial ROS (MitoSox; A, n = 5, 5, 5; C, n = 8, 8, 8; G, n = 8, 8, 8), mitochondrial membrane potential (MitoRed; B, n = 8, 8, 8; D, n = 20, 20, 20; H, n = 8, 8, 8), mitochondrial calcium (Ca2+; Rhod2; E, n = 8, 8, 8; F, n = 8, 8, 8) in the indicated groups. For studies of MitoSox and MitoRed, recombinant FXa protein (10nM) was administered for a total of 3 h and other compounds were administered for 1 h before the administration of FXa at the following concentrations: PAR1 inhibitor (PAR1-i), 1μM; MitoTEMPO, 10μM; MCU inhibitor (250nM). For studies of Rhod2, recombinant FXa was administered for a total of 30 min, with or without the reagents described above.

(I) Results of qPCR for expression of coagulation factor VII (F7; n = 5, 6), factor X (F10; n = 6, 6), tissue factor (F3; n = 4, 5), and F2r (PAR-1 transcript; n = 6, 6) in differentiated brown adipocytes incubated with control adenovirus (Mock) or adenovirus encoding constitutively activated Hif-1a (Ad-Hif-1a; 10 MOI, 48 h for F7, F10, and F2r; 30 MOI, 24 h for F3).

(J) Enzyme-linked immunosorbent assay (ELISA) for tissue factor (n = 10, 10), coagulation factor VII (FVII; n = 9, 14), and FXa (n = 11, 9) in conditioned medium from differentiated brown adipocytes incubated with control adenovirus (Mock) or Ad-Hif-1a.

(K and L) MitoSox/MitoGreen ratio (K, n = 8, 7, 7) and MitoRed/MitoGreen ratio (L, n = 8, 8, 8) in differentiated brown adipocytes incubated with conditioned medium (CM) obtained from brown adipocytes infected with Mock (Mock-CM), Ad-Hif-1a (Ad-Hif-1a-CM), or Ad-Hif-1a-CM + PAR1-i. Data were analyzed by a 2-tailed Student’s t test (I-L), or by 2-way ANOVA followed by Tukey’s multiple comparison test (A and C-H), or non-parametric Kruskal Wallis test (B). ∗p < 0.05, ∗∗p < 0.01. Values represent the mean ± SEM NS = not significant. All data are from different biological replicates. See also Figure S6.