Table 1.
Serum biochemical parameters of human PSC patients.
Fig 1.
Increased expression of autophagic proteins in PSC.
Western blotting of p62, ATG5/12, ATG7, Beclin1, LC3B, LAMP1 and LAMP2 in liver extracts prepared from patients with PSC and Controls. Blots were normalized to GAPDH expression. Data are presented as Mean ± STDEV and were statistically analyzed using Student’s t-test, *p<0.05, **p<0.01, (N = 6/group).
Fig 2.
Increased expression of autophagic proteins in acute murine cholestasis.
A. qPCR analysis of mRNA for autophagic proteins in liver tissue isolated from 3-day sham and BDL adult mice (N = 5/group). Expression was normalized against HPRT. B. Western analysis of autophagic proteins in liver tissue isolated from BDL mice (N = 5/group). Blots were normalized to GAPDH expression. C. qPCR analysis of mRNA for autophagic proteins in liver tissue isolated from 10-week old Mdr2KO mice (N = 4/group). D. Western analysis of autophagic proteins in liver tissue isolated from 10 week on Mdr2KO mice (N = 6/group). Blots were normalized to GAPDH expression Data are presented as Mean ± STDEV and were statistically analyzed using a Student’s t-test, *p<0.05, **p<0.01, ***P<0.001, ****p<0.0001, N = 6/group.
Fig 3.
Increased accumulation of p62, LC3b, LAMP1 and LAMP2 in PSC and BDL mice is located primarily in parenchymal cells that surround the portal triad.
Tissue sections from normal human liver, PSC, sham and BDL mice were examined for p62, LC3b, LAMP1 and LAMP2 expression. A. Human control and PSC. B. Sham and BDL. N = 3-5/group, 200X, CV-central vein, PT-portal triad. Arrows indicate increased staining. C. 10-week old WT and Mdr2KO (200X). N = 3-5/group, 200X, CV-central vein, PT-portal triad. Arrows indicate increased staining.
Fig 4.
Colocalization of p62 in macrophages during cholestasis.
A. Colocalization of p62 and CD68+ macrophages in human PSC. Paraffin embedded formalin fixed tissue sections from normal human PSC liver were analyzed immunohistochemically using a rabbit polyclonal antibody directed against p62, a mouse monoclonal antibody directed against the macrophage marker CD68 followed by FITC 488 conjugated anti-rabbit and Alexa Fluor 594-conjugated anti-mouse secondary antibodies. Figures are representative of hepatic tissue isolated from three Control and four PSC patients respectively, (200X). B-C. Colocalization of p62 and F4/80 positive macrophages in murine cholestasis. Paraffin embedded formalin fixed tissue sections from B. Sham, BDL liver C. WT, Mdr2KO liver were analyzed immunohistochemically using a rabbit polyclonal antibody directed against p62, a rat polyclonal antibody directed against the macrophage marker F4/80 followed by FITC 647 conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-rat secondary antibodies. Slides were examined using fluorescent microscopy (Blue arrows = p62 positive hepatocytes, red arrows = macrophages, yellow arrows-colocalization), nuclei were visualized by DAPI. Figures are representative of hepatic tissue from three each of sham, WT and the BDL, Mdr2KO animals, respectively (200X).
Fig 5.
Increased periportal oxidative injury in acute cholestasis.
Tissue sections from sham and BDL mice were probed immunohistochemically for pH2Ax and 4-HNE. N = 4/group, 200X, PT-portal triad, CV-Central vein. Arrows indicate area of increased staining.
Fig 6.
Increased periportal nuclear localization of Nrf2 in human and murine cholestasis.
Tissue sections were examined for Nrf2 nuclear localization A. Normal human liver, PSC (200X). B. Sham mice and BDL mice (100X). C. Accumulation of ubiquitinated proteins in parenchymal cells surrounding the injured portal triad in PSC. n = 3-4/group, (200X). CV-central vein, PT-portal triad. Arrows indicate increased periportal staining.
Fig 7.
Increased periportal oxidative stress and upregulation of Nrf2 antioxidant proteins in acute cholestasis.
A. Western analysis of selected Nrf2 target proteins (HO-1, GCLC, CBR3, GSTμ, NQO1) in liver tissue isolated from sham and BDL mice. Blots were normalized to GAPDH expression. Data are presented as Mean ± SEM and were statistically analyzed using a student’s t-test, *p<0.05, **p<0.01, ***P<0.001, ****p<0.0001, N = 5/group. B. Tissue sections from sham and BDL mice were probed immunohistochemically for GSTμ, Cbr3 and HO-1. N = 4/group, 200X, PT-portal triad, CV-Central vein. Arrows indicate area of increased staining.
Fig 8.
Colocalization of p62 and 4-HNE in liver in cholestasis A. Control and PSC liver (200X). B. Sham and BDL cholestatic liver (100X) C. WT and Mdr2KO liver (100X). Arrows indicate periportal hepatocytes of increased staining/colocalization. n = 4/group, Blue-Dapi, Green-4-HNE, Cyan-p62. CV-central vein, PT-portal triad.
Fig 9.
p62 is a target of reactive aldehydes in in vitro cell culture and human PSC.
Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by streptavidin bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A. RAW264.7, B. HepG2. C. Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.
Table 2.
4-HNE modified peptides identified from tryptic digests of 4-HNE-treated recombinant p62.