Figures & data
Figure 1. 4-PBA reduced RFP-induced toxicity and apoptosis of HepG2 cells.
(a) HepG2 cells were treated by RFP (50 uM) and/or 4-PBA (0.1 mM, 0.5 mM, 1 mM) for 48 h. Cell viability was detected via CCK-8 assay. (b) HepG2 cells were treated by RFP (50 uM) and/or 4-PBA (1 mM) for 48 h. Cell apoptosis was checked by PI/Annexin V double staining. ***P < 0.001 vs. control group; #P< 0.05, ##P< 0.01, ###P< 0.001 vs. RFP group; &P< 0.05, &&P< 0.01 vs. 4-PBA group.
Figure 2. 4-PBA attenuated the suppression of MRP2 by RFP.
HepG2 cells were treated by RFP (50 uM) and/or 4-PBA (1 mM) for 48 h. (a and b) mRNA and protein expression of MRP2 were detected through qRT-PCR assay and Western blotting, respectively. (c) Immunofluorescence assay was carried out to further test MRP2 expression. (d) The ubiquitination of MRP2 was checked by in vitro ubiquitination assay. (e) The protein level of GP78 was evaluated via Western blotting. ***P< 0.001 vs. control group; #P< 0.05, ###P< 0.001 vs. RFP group; &P< 0.05, &&P< 0.01 vs. 4-PBA group.
Figure 3. 4-PBA improved RFP-induced intracellular calcium disorder and ER stress response.
HepG2 cells were treated by RFP (50 uM) and/or 4-PBA (1 mM) for 48 h. (a) The intracellular calcium concentration was tested via fluo-3/AM staining. (b) The protein levels of GRP78 BIP, CHOP, Cleaved-Caspase-12, Cleaved-Caspase-3, PERK, ATF6, XBP-1, IRE-1, p-JNK and p-p38 in cells were evaluated via Western blotting. **P< 0.01, ***P< 0.001 vs. control group; #P< 0.05, ##P< 0.01, ###P< 0.001 vs. RFP group; &P< 0.05, &&P< 0.01 vs. 4-PBA group.
Figure 4. 4-PBA alleviated RFP-caused increase of AP2 and clathrin.
(a and b) HepG2 cells were treated by RFP (50 uM) and/or 4-PBA (1 mM) for 48 h. The mRNA and protein expressions of AP2 and clathrin in cells were tested via qRT-PCR and Western blotting. (c and d) Following CHOP-siRNA (or CHOP-plasmid) transfection and/or RFP (50 uM) or 4-PBA (1 mM) treatment for 48 h, the CHOP mRNA expression was tested via qRT-PCR assay and the protein expressions of AP2 and clathrin in HepG2 cells were tested via Western blotting. *P< 0.05, **P< 0.01, ***P< 0.001 vs. control group; #P< 0.05, ##P< 0.01, ###P< 0.001 vs. RFP group; &P< 0.05, &&&P< 0.001 vs. 4-PBA group.
Figure 5. 4-PBA protected mice against RFP-induced cholestatic liver injury.
RFP-induced cholestasis mouse model was established and treated by 4-PBA. (a) Serum bilirubin concentration was tested vi ELISA. (b) The protein expression of MRP2 in liver tissues was measured via Western blotting. (c) The ubiquitination of MRP2 in liver tissues was tested via ubiquitination assay. (d) The protein levels of GRP78 BIP, CHOP, Cleaved-Caspase-12, Cleaved-Caspase-3, PERK, ATF6, XBP-1, IRE-1, p-JNK and p-p38 in liver tissues were evaluated via Western blotting. *P< 0.05, **P< 0.01, ***P< 0.001 vs. control group; #P< 0.05, ##P< 0.01 vs. RFP group.
