Fig 1.
The expression level of PDIA3 was increased in fat loaded cell models of NAFLD.
(A) Oil red O staining. Original magnification, 200×. (B) Intracellular triglyceride assay. **P<0.01. (C) Western blot analysis of PDIA3 protein expression. (D) The relative expression level quantified by densitometry analysis of bands and normalized to GAPDH protein. All studies were conducted at least three times in triplicate, *P<0.05.
Fig 2.
PDIA3 siRNA interference effect.
(A) PDIA3 siRNA markedly reduces the PDIA3 protein level in L02 cells. (B) Relative protein level of PDIA3 after siRNA transfection. The PDIA3-s1 has the most significant knockdown effect. **P<0.01 versus negative siRNA transfected cells. Three independent experiments were performed.
Fig 3.
PDIA3 siRNA exacerbates hepatocellular steatosis in L02 cells.
(A) Oil red O staining of cells treats with or without FFA after transfection of PDIA3-s1 or PDIA3-neg. Original magnification, 200×. (B) Influence of PDIA3 knockdown on intracellular triglyceride level induced by FFA. These experiments were repeated three times. *P<0.05.
Fig 4.
PDIA3 siRNA aggravates cellular apoptosis of steatotic L02 cells.
(A) Flow cytometry scatter plot after Annexin V-FITC/PI staining: left lower quadrant: normal cells; right lower quadrant: early apoptosis cells (FITC Annexin V positive and PI negative); right upper quadrant: late apoptosis cells (both FITC Annexin V and PI positive); left upper quadrant: necrotic cells. The percentage represents the proportion of cells in each quadrant. (B) The histogram of early, late and total apoptosis after three independent experiments. **P<0.01.
Fig 5.
The expression level of lipid synthesis proteins.
(A) Western blot analysis of PDIA3, FAS, SREBP1 and ACC1 proteins expression. (B.C.D.E) The relative expression levels quantified by densitometry analysis of bands and normalized to GAPDH protein. These experiments were repeated three times in triplicate. *P<0.05, **P<0.01.
Fig 6.
The expression level of ERS and apoptosis related proteins.
(A) Western blot analysis of GRP78, PERK, p-PERK, ATF-6α, IRE1, p-IRE1 and CHOP proteins expression. (B.C.D.E.F) The relative expression levels quantified by densitometry analysis of bands and normalized to GAPDH protein. These experiments were repeated three times in triplicate. *P<0.05, **P<0.01.
Fig 7.
The schematic diagram illustrating the potential role of PDIA3 in the FFA-induced hepatocyte steatosis and apoptosis in L02 cells.