Fig 2: Alcohol induces ABL2-mediated PPARγ via HIF1α. (A) Gene set enrichment analysis plot of HIF1 signaling. (B) FPKM data of HIF1 signaling genes (n = 3 technical replicates per condition). (C) Western blot analysis of HIF1α in liver tissue from NIAAA 10d + 1b AlbCre and ABL2 KO pair- and alcohol-fed mice (n = 2 mice per group). (D) Western blot analysis of shScr and shABL2 HepG2 cells cultured in absence and presence of 50 mmol/L alcohol for 48 hours. Data are representative of 3 biological replicate experiments. Western blot analysis of vehicle (DMSO). (E) GMB475- and (F) nilotinib-treated HepG2 cells cultured in absence and presence of 50 mmol/L alcohol for 48 hours. Data are representative of 2 biological replicate experiments. (G) Western blot analysis of HepG2 cells treated with vehicle (DMSO) or 25 μmol/L PX478 for 48 hours in absence and presence of 50 mmol/L alcohol. Data are representative of 3 biological replicate experiments. (H) Western blot analysis of HepG2 cells treated with vehicle (H2O) or 50 mmol/L cobalt chloride for 48 hours. Data are representative of 3 biological replicates. (I) Western blot and (J) qPCR analysis of stable control (PLX-Luc) and HIF1A-overexpressing HepG2 cells after 5 days of 2 μg/mL blasticidin selection. Data are representative of 2 biological replicate experiments. Statistical analysis conducted by one-way ANOVA test. (K) Western blot analysis of vehicle (DMSO) or GMB475-treated mouse primary hepatocytes cultured in absence and presence of 50 mmol/L alcohol for 48 hours. Data are representative from 2 independent experiments with biological duplicates. (L) Oil Red staining (original magnification, ×100) of vehicle (DMSO) or GMB475-treated mouse primary hepatocytes cultured in absence and presence of 50 mmol/L alcohol for 48 hours with accompanying (M) quantification. Data are representative from 2 independent experiments with biological duplicates. Statistical analysis conducted by one-way ANOVA test. Values are mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Fig 3: Deletion of Abl2 in hepatocytes does not affect morphology, histology, proliferation, or apoptosis in mouse liver. (A) Photographs of livers and representative pictures of H&E, Ki67 immunohistochemistry, and TUNEL from AlbCre and AlbCre; Abl2flox/flox mice at 6–8 weeks of age. (B) Quantification of Ki67 staining (n = 3). (C) Quantification of TUNEL staining (n = 3). Statistical analysis conducted by Student t test. Values are mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Fig 4: Alcohol promotes ABL2 activation and PPARγ expression in ABL2-dependent manner in vitro. (A) Western blot analysis of HepG2 cells cultured in absence and presence of increasing alcohol concentrations for 72 hours. Data are representative of 3 independent experiments. (B) Western blot analysis of HepG2 cells transiently transfected with control (PLX304-FLAG) and ABL2-overexpressing vectors (PLX304-ABL2) for 48 hours. Data are representative of 3 independent experiments. (C) Western blot and (D) qPCR analysis of stable control (shScr) and ABL2 knockdown (shABL2-3 and shABL2-4) HepG2 cells. Statistical analysis conducted by one-way ANOVA test. (E) qPCR analysis of Pparg mRNA expression and of shScr control and shABL2 knockdown cells cultured in absence and presence of 50 mmol/L alcohol for 48 hours. Data are representative of 3 independent experiments. Statistical analysis conducted by one-way ANOVA test. (F) Western blot analysis of shScr control and shABL2 knockdown cells cultured in absence and presence of 50 mmol/L alcohol for 48 hours. Data are representative of 3 independent experiments. (G) Oil Red O staining (original magnification, ×100) with (H) quantification of control (shScr) and ABL2 knockdown cells cultured in absence and presence of 50 mmol/L alcohol for 48 hours. Data are representative of 3 independent experiments. Statistical analysis conducted by one-way ANOVA test. (I) Venn diagram of differentially expressed genes among shScr and shABL2 cells treated without and with 50 mmol/L alcohol for 72 hours (n = 3 technical replicates per condition). (J) Top down-regulated KEGG pathways between ABL2 knockdown and shScr cells cultured in absence and presence of 50 mmol/L alcohol for 72 hours (n = 3 technical replicates per condition). (K) PPAR signaling gene set enrichment analysis plot of shABL2 and shScr cultured in absence and presence of 50 mmol/L alcohol for 72 hours. (L) Fragments per kilobase of transcript per million mapped reads (FPKM) data of PPAR signaling genes (n = 3 technical replicates per gene). Statistical analysis conducted by one-way ANOVA test. (M) FPKM data of PPAR isoforms. Statistical analysis conducted by one-way ANOVA test. (N) Western blot analysis of HepG2 cells treated with vehicle (DMSO) or 2.5 μmol/L ABL kinase inhibitors in absence and presence of 50 mmol/L alcohol for 48 hours. (O) Oil Red O staining of HepG2 cells treated with vehicle (DMSO) or 2.5 μmol/L GMB475 in absence and presence of 50 mmol/L alcohol (original magnification, ×100). Results are representative of 3+ biological replicate experiments. Values are mean ± standard errors of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Fig 5: Hepatic deletion of Abl2 suppresses alcohol-induced PPAR signaling and PPARγ. (A) Serum EtOH levels from AlbCre control and ABL2 KO mice with both pair-fed and alcohol-fed groups (n = 5). Statistical analysis conducted by one-way ANOVA test. (B) Volcano plot and (C) top 10 down-regulated KEGG pathways from RNASeq analysis of ABL2 KO and WT NIAAA 10d + 1b alcohol-fed mice (n = 3 mice per group). Equal males and females were used for each group. (D) Western blot analysis of liver tissue from healthy donors and alcoholic hepatitis patients. (E) Western blot analysis of liver tissue from NIAAA 10d + 1b AlbCre and ABL2 KO pair- and alcohol-fed mice (n = 3 mice per group). (F) Western blot analysis of liver tissue from NIAAA 10d + 1b AlbCre and ABL1 KO pair- and alcohol-fed mice (n = 2 mice per group). qPCR analysis of (G) Pparg, (H) Plin2, and (I) Fsp27 mRNA expression of NIAAA 10d + 1b AlbCre and ABL2 KO pair- and alcohol-fed mouse liver tissue (n = 5 mice per group). Statistical analysis conducted by one-way ANOVA test. Values are mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.