Table 2. List of antibodies
Antibody Manufacturer Catalog
Number Dilution Fold Application Primary Antibodies
Anti-CK19 Santa Cruz
Biotechnology sc-33119 1/5000
(in 4% skim milk) WB Anti-CK19 Santa Cruz
Biotechnology sc-33119 1/100 IF
Anti-EpCAM BD Bioscience 552370 1/300 IF
Anti-FLAG Sigma-Aldrich F3165 1/1000
(in 4% skim milk) WB
Anti-FLAG Sigma-Aldrich F3165 1/200 ChIP
Anti-HNF4α Santa Cruz
Anti-α-Tubulin GeneTex GTX112141 1/10000 WB
Normal Rabbit IgG
Santa Cruz
Biotechnology sc-2027x 1/400 ChIP
Secondary Antibodies Anti-goat
AF488
Life
Technologies A11055 1/800 IF
Anti-goat
HRP R&D System HAF019 1/10000 WB
Anti-mouse HRP
Jackson
ImmunoResearch 115-035-003 1/10000 WB Anti-rat
AF488
Life
Technologies A21208 1/800 IF
Anti-rabbit HRP
Jackson
ImmunoResearch 111-035-003 1/10000 WB WB: Western Blotting; IF: Immunofluorescence staining; ChIP: Chromatin Immunoprecipitation; AF: Alexa Fluoro
Figure 1. Confirmation of HIF-1α protein expression
Western blot analysis of HIF-1α expression in primary hepatocytes cultured under normoxia or hypoxia for 48 hours. CBR staining served as loading control.
Figure 2. Confirmation of hypoxic condition in primary hepatocyte by HIF-1α target
gene expression
qRTPCR analysis of EPO mRNA isolated from primary hepatocytes cultured under normoxia or hypoxia for 48 hours. Results were normalized using GAPDH, Eef2 and Rplp0 as reference genes. ***p < 0.001, n = 3; Student’s t-test.
Figure 3. Hypoxia decreased the expression of mature hepatocyte marker in primary
hepatocyte
qRTPCR analysis of HNF4α mRNA isolated from primary hepatocytes cultured under normoxia or hypoxia for 48 hours. Results were normalized using GAPDH, Eef2 and Rplp0 as reference genes. **p < 0.01, n = 3; Student’s t-test.
Figure 4. Hypoxia increased the expression of LPC marker in primary hepatocyte
qRTPCR analysis of EpCAM mRNA isolated from primary hepatocytes cultured under normoxia or hypoxia for 48 hours. Results were normalized using GAPDH and β-actin as reference genes. **p < 0.01, n = 3; Student’s t-test.
Figure 5. Hypoxia increased the expression of stemness marker in primary
hepatocyte
qRTPCR analysis of Sox2 and Nanog mRNA isolated from primary hepatocytes cultured under normoxia or hypoxia for 48 hours. Results were normalized using GAPDH, Eef2 and Rplp0 as reference genes. **p < 0.01, n = 3; Student’s t-test.
Figure 6. Hypoxia altered the expression of mature hepatocyte marker and LPC
marker in primary hepatocyte
Western blot analysis of HNF4α and CK19 expression in primary hepatocytes cultured under normoxia or hypoxia for 48 hours. CBR staining served as loading control.
Figure 7. Hypoxia increased EpCAM and CK19 expression in primary hepatocyte
Immunofluorescence staining of EpCAM and CK19 expression in primary hepatocytes cultured under normoxia or hypoxia for 48 hours. Images were captured and analyzed by IN Cell Analyzer 2000. Fluorescence intensities of each cell were plotted as histogram.
Figure 8. HIF-1α enhances EpCAM transcription activity in HEK293T and Huh7
(A) Scheme of EpCAM promoter constructed in pGL4.10[luc2]. DNA fragment
extending from -895 bp to 75 bp relative to the transcription start site of EpCAM was inserted into pGL4.10[luc2].
(B) Luciferase intensities of HEK293T and Huh7 transfected with pGL4-EpCAM
promoter and cultured under normoxia or hypoxia for 48 hours. pcDNA-HIF-1α addition
was showed on the figure. Luciferase intensities were normalized by RFP intensities. *p
< 0.05, **p < 0.01, ***p < 0.001, n = 3; two-way ANOVA followed by Tukey’s multiple comparisons test.
Figure 9. HIF-1α directly binds onto EpCAM promoter
ChIP-qRTPCR analysis of HEK293T transfected with pcDNA-2FLAG-HIF and cultured under normoxia or hypoxia for 48 hours.
(A) Western blot analysis of HIF-1α expression using anti-FLAG antibody. α-Tubulin
served as loading control.
(B) Fragments of EpCAM promoter were detected more by anti-FLAG antibody
immunoprecipitation than IgG. n = 3; Student’s t-test.
Figure 10. Hypoxia increased the expression of Wnt signaling target genes in
primary hepatocyte in primary hepatocyte
qRTPCR analysis of Axin2 and Lef1 mRNA isolated from primary hepatocytes cultured under normoxia or hypoxia for 48 hours. Results were normalized using GAPDH, Eef2 and Rplp0 as reference genes. **p < 0.01, ***p < 0.001, n = 3; Student’s t-test.
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