Nucleic Acids Research, gkac070, https://doi.org/10.1093/nar/gkac070
Errors were introduced in Figures 2A, 3A, 5BC and 6AB which all lack the densitometry scanning data of Western, Northern and Southern blots. The original published article has been updated with the correct figures.
Figure 2.
Cis-element ‘ATTGG’ upregulates EII/Cp promoter activity by recruiting HMGA1. Huh7 cells cultured in 24-well plate were co-transfected with 0.4 μg of pCp or pCpm reporter, 0.4 μg of shHMGA1-expressing (pshHMGA1) or control plasmids (pshCtrl) (A), or Flag-tagged HMGA1 expression (pHMGA1-Flag) or control plasmids (pCtrl) (B), and 0.1 μg of pRL-TK. At 48 h post-transfection, relative promoter activities were measured using dual-luciferase reporter assay. HMGA1 and HMGA1-Flag protein levels were determined in western blot using HMGA1 and Flag antibodies, respectively, and numbers denote normalized densitometry scanning data. Group means and SEMs of normalized firefly versus Renilla luciferase activity ratios within transfection group were presented and significances calculated using unpaired two-tailed t-test; *, P < 0.05; ***, P < 0.001. Intracellular distribution of endogenous HMGA1 (C) and exogenous HMGA1-Flag (D) were analyzed using immunofluorescence and nucleocytoplasmic separation test coupled with western blot. Lamin A/C and annexin A1 (ANXA1) were used as markers of nuclear and cytoplasmic fractions, respectively. (E) Biotinylated Cp and Cpm probes (nt -32 to -10 of Cp) used in DNA-pulldown assay. Chemically synthesized probes were added to nuclear protein extracts (S1 and S2) and incubated with streptavidin-coupled beads. After washing, beads were subjected to western blot (F).
Figure 3.
HMGA1 upregulates EII/Cp promoter activity by recruiting FOXO3α and PGC1α to ‘ATTGG’ element. (A) Huh7 cells cultured in 24-well plate were co-transfected with 0.4 μg of pCp or pCpm reporter, 0.4 μg of FOXO3α, PGC1α or control shRNA-expressing plasmids, and 0.1 μg of pRL-TK. At 48 h post-transfection, relative promoter activities were measured using dual-luciferase reporter assay. FOXO3α and PGC1α protein levels were determined in western blot and numbers denote normalized densitometry scanning data. Group means and SEMs of normalized firefly versus Renilla luciferase activity ratios within transfection group were presented and significances calculated using unpaired two-tailed t-test; *, P < 0.05; **, P < 0.01. Intracellular distribution of endogenous FOXO3α and PGC1α were analyzed using immunofluorescence (B) and nucleocytoplasmic separation test coupled with western blot (C). Lamin A/C and annexin A1 (ANXA1) were used as markers of nuclear and cytoplasmic fractions, respectively. (D) Co-immunoprecipitation of endogenous FOXO3α and PGC1α with endogenous HMGA1. (E) Endogenous FOXO3α and PGC1α, in addition to HMGA1, were pulled down by biotinylated Cp and Cpm probes (nt -32 to -10 of Cp) as shown in Figure 2E. Chemically synthesized probes were added to nuclear protein extracts (S1 and S2) and incubated with streptavidin-coupled beads. After washing, beads were subjected to western blot.
Figure 5.
HMGA1 promotes HBV gene expression and replication in HBV-infected HepG2/NTCP cells. HepG2/NTCP cells were infected with HBV virions at a MOI of 1000 geq/cell overnight. Culture media were changed the next day, and at day 2, cells were transduced by shHMGA1-expressing lentivirus or control virus (Lenti-shHMAG1 or Lenti-shCtrl) (A and B), or Lenti-HMGA1-Flag or control virus Lenti-Ctrl (A and C). Culture media were changed at day 3, and then every 2 days. At day 7, intracellular HBV RNA, core-associated DNA and cccDNA were examined using northern and southern blot, and qrtPCR, respectively. Endogenous and exogenous HMGA1 were determined in western blot using anti-HMGA1 and anti-Flag antibodies, respectively. HBsAg and HBeAg in culture media were assayed using ELISA. The relative levels of HBV RNA and DNA were quantified using densitometry scanning. Group means and SEMs of normalized values within transfection group were presented. Data of Lenti-shHMGA1or Lenti-HMGA1-Flag-transduced groups were compared against control virus group using unpaired two-tailed t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 6.
HBV upregulates HMGA1 expression via HBx. (A) Endogenous HMGA1 in HepG2/NTCP and primary human hepatocytes (PHH) cells with or without HBV infection were analyzed in western blot. (B) Huh7 cells were transfected with HBV replicon plasmid p1.3HBV, or p1.3HBV with HBx obliterated (p1.3HBV/Xnull), or control vector. At day 2 post transfection, intracellular HMGA1 was analyzed in western blot. (C) Huh7 cells were transfected with p1.3HBV, Flag-tagged HBx expression plasmid pHBx-Flag or control vector. At day 2 post transfection, HMGA1 mRNA levels were measured using RT-qrtPCR. (D) HMGA1 promoter reporter plasmid and pRL-TK were co-transfected with control vector, p1.3HBV or pHBx-Flag into Huh7 cells. At day 2 post transfection, cells were lysed and promoter activities were measured using dual-luciferase reporter assay. (E) Huh7 cells transfected with pHBx-Flag or control vector were subjected to co-immunoprecipitation analysis using anti-Flag antibody. (F) Endogenous SP1 and exogenous Flag-tagged HBx were pulled down by biotinylated probes encompassing the three SP1 binding sites in HMGA1 promoter (Probes 1–3) or corresponding mutant probes with SP1 binding sites mutated (Probes 1m-3m) as shown in Supplementary Table S4. Chemically synthesized probes were added to nuclear protein extracts (S1-6) and incubated with streptavidin-coupled beads. (G) In vitro translated SP1 and HBx-Flag were incubated with a mixture of biotinylated wild-type or mutant SP1 binding site probes and streptavidin-coupled beads. After washing, beads were subjected to western blot. Numbers denote normalized densitometry scanning data (A and B). Group means and SEMs of normalized values within transfection group were presented and significances calculated using unpaired two-tailed t-test; ***, P < 0.001.
The corrections do not affect the results, discussion or conclusions presented in the article.
The publisher apologises to the authors and readers for these errors.