Fig 1: Silencing of BMP6 in Huh7 cells does not affect APAP-induced cell death. Experimental conditions (A–E): siBMP6 and control (siC) Huh7 cells treated with 20 mM APAP for 16 h (N > 3 independent experiments). A BMP6 mRNA levels determined by RT-qPCR and normalized to 36B4 gene expression. Data are expressed as fold of change relative to control condition (siC-C). B Representative blots of the cell lysates with the indicated antibodies and the corresponding quantification. Data are expressed as fold of change relative to control condition (siC). C Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (C, 100%). D Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). E Representative blots of the cell lysates with the indicated antibodies. F Experimental conditions (F–I): siBMP6 and control (siC) Huh7 cells treated with 20 mM APAP for 16 h. Huh7 cells incubated with siBMP6- or siC-CM for 24 h (N > 3 independent experiments). G Representative blot of the cultured media (CM) with BMP6 antibody. Ponceau staining was used as loading control. H. Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (C, 100%). I Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). *p < 0.05, ***p < 0.005 and ****p < 0.0001, APAP vs. C; #p < 0.05 and ##p < 0.01, siBMP6 vs. siC
Fig 2: Huh7 hepatocytes release BMP6 after APAP exposure. A Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (100%). B Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). C Representative 40X images of DAPI staining. D ROS production detected with DHE probe represented as DHE fluorescence F/F0 (a.u.). E Representative blots of the cell lysates with the indicated antibodies. F BMP6 mRNA levels determined by RT-qPCR and normalized to 36B4 gene expression. Data are expressed as fold of change relative to control condition. G Representative blot of the cultured media (CM) with BMP6 antibody. Ponceau staining was used as loading control. Experimental conditions: Huh7 treated with 10 mM and 20 mM APAP for 2 (D), 6 (E) or 16 (A, B, C, F, G) hours (N > 3 independent experiments). ****p < 0.0001, 10 or 20 mM APAP vs. non-treated cells
Fig 3: Circulating BMP6 is increased in mice after APAP overdose and in patients with APAP intoxication. Experimental conditions: Mice i.p. injected with vehicle (saline) or APAP (300 mg/kg) and sacrificed 6 or 24 h after APAP administration (n = 12 animals per group). A Serum levels of BMP6 determined by ELISA. Data are expressed as pg/ml. B and C Correlation of serum BMP6 levels with hepatic expression of BMP6, circulating ALT and AST, respectively. Study population: 18 patients with APAP overdose, 9 with DILI (ALT > 100 U/L) and 9 without DILI. D Serum levels of BMP6 determined by ELISA. Data are expressed as pg/ml. E Correlation of serum BMP6 levels with circulating ALT levels. **p < 0.01 and ***p < 0.005, APAP 6 h or 24 h vs. Control
Fig 4: Immortalized mouse hepatocytes showed elevated expression and release of BMP6 after APAP treatment. A Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (100%). B Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). C Representative 40X images of DAPI staining. D ROS production detected with DHE probe represented as DHE fluorescence F/F0 (a.u.). E Representative blots of the cell lysates with the indicated antibodies. F Bmp6 mRNA levels determined by RT-qPCR and normalized to 36b4 gene expression. Data are expressed as fold of change relative to control condition. G Representative blot of the cultured media (CM) with BMP6 antibody. Ponceau staining was used as loading control. Experimental conditions: immortalized mouse hepatocytes treated with 1 mM or 5 mM APAP for 2 (D), 6 (E) or 16 (A, B, C, F, G) hours (N > 3 independent experiments). **p < 0.01, ***p < 0.005 and ****p < 0.0001, 1 or 5 mM APAP vs. non-treated cells
Fig 5: Hepatic BMP6 expression is increased in an animal model of APAP-induced ALF. A Representative 4X and 10X images of H&E and percentage of necrotic area quantification (%). B ALT and AST activity determination in serum samples. C and D Hmox1 and Bmp6 mRNA levels determined by RT-qPCR and normalized to 36b4 gene expression. Data are expressed as fold of change relative to control condition (Control). E Correlation of Bmp6 mRNA expression with Hmox1 mRNA expression. F Representative 10X and 20X images of BMP6 immunostaining from liver sections and its quantification. Data are expressed as arbitrary units (a.u.). G Correlation of BMP6 hepatic expression with percentage of necrotic area. Experimental conditions: Mice i.p. injected with vehicle (saline) or APAP (300 mg/kg) and sacrificed 6 or 24 h after APAP administration (n = 12 animals per group). **p < 0.01, ***p < 0.005 and ****p < 0.0001, APAP 6 h or 24 h vs. Control
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