Figure 3. Multiple routes lead to MiTF expression.

(A and B) Relative MiTF (A) or pSMAD2/3 (B) levels evaluated by FACS in indicated cell lines. Data are shown as mean ± SEM. (A) n = 14 (HSC93, HSC99), n = 4 (HSC72, HSC536). (B) n = 6 (HSC93, HSC99), n = 4 (HSC72), and n = 3 (HSC536). (C and D) Western blots illustrating MiTF, pSMAD2/3, and p-p38 expression in extracts from WT, FANCA^–/–, or FANCC^–/– cells, treated (6 hours) or not treated with 10 ng/mL of TGF-β, activin A, or TNF-α. Vertical lines (C) separate noncontiguous samples migrated on the same gel. Vertical lines in D were added to improve readability. (E) Western blots showing MiTF and p-p38 expression in FANCA^–/– or FANCC^–/– cells treated with the p-p38 inhibitor SB203580. β-Actin was used as a loading control. (F) TNF-α concentration (pg/mL) measured by ELISA in supernatants from indicated WT or FANC pathway–deficient cells 48 hours after seeding. Data are shown as mean ± SEM (n = 5). (G) Relative MiTF expression evaluated by qRT-PCR in FANCA^–/– cells (HSC99) 6 hours following exposure to 10 ng/mL TGF-β, activin A, or TNF-α. Data are shown as mean ± SEM (n = 3). (H) Relative MiTF expression evaluated by qRT-PCR in FANCA^–/– lymphoblasts treated for 2 hours with DMSO, SB203580 (10 μM), BIRB-796 (10 μM), SIS3 (2.5 μM), SD208 (10 μM), curcumin (50 μM), LY294002 (10 μM), and NBD (1 μM). The results were normalized against Actin expression and calculated relative to untreated cells (n = 5 [NT, DMSO, SB203580, BIRB-796, SIS3, SD208, SB203580 + SIS3], n = 4 [curcumin], and n = 3 [LY294002 and NBD]). (I) Western blots showing MiTF and pAKT expression in WT and FANC pathway–deficient cells. β-Actin was used as a loading control. (J) Summary of our previous observations combined with data from the literature. Statistical significance was assessed using an unpaired 2-tailed t test with Welch’s correction (F) or 1-way ANOVA with Dunnett’s correction (A, B, G, and H). *P < 0.05; **P < 0.01; ***P < 0.001.