CELL BIOLOGY Correction for “HERES, a lncRNA that regulates canonical and noncanonical Wnt signaling pathways via interaction with EZH2,” by Bo-Hyun You, Jung-Ho Yoon, Hoin Kang, Eun Kyung Lee, Sang Kil Lee, and Jin-Wu Nam, which was first published November 15, 2019; 10.1073/pnas.1912126116 (Proc. Natl. Acad. Sci. U.S.A. 116, 24620–24629).
The authors note, “Due to an error during figure preparations, the Western blot images for β-actin in Figs. 3E, 4H, 5C, and 6H appeared incorrectly. We are very sorry for any inconvenience this Correction may cause.” The full corrected Figs. 3, 4, 5, and 6 appear below with their legends. These errors do not affect the conclusions of the article.
Fig. 3.
HERES modulates cell proliferation, migration, invasion, and colony formation. (A) Cell viability was measured using an MTS assay in KYSE-30 and HCE-7 cells transfected with siControl (NC), siHERES (si_1 and si_2), or siHERES followed by pcDNA-HERES (si_1+pcDNA, si_2+pcDNA). Growth curves were compared between siHERES- and siControl-transfected cells, and between pcDNA-HERES+siHERES- and siHERES-transfected cells. Wound healing assays (B), invasion assays (C), and colony formation assays (D) were performed in KYSE-30 and HCE-7 cells after HERES knockdown (all assay images at 4× magnification). The bar graphs represent the frequency of wound closure (B) and the number of invading cells (C) and colonies formed (D). Data represent the mean ± SD from 3 independent experiments (A–D). (E) Expression of EMT markers in KYSE-30 and HCE-7 cells transfected with siControl or the indicated combinations of siHERES and pcDNA-HERES as determined by immunoblot. *P ≤ 0.05, **P ≤ 0.01.
Fig. 4.
HERES epigenetically regulates genes involved in canonical and noncanonical Wnt signaling pathways. (A) Changes in the expression of cancer-related genes in response to siHERES treatment compared to siControl are shown. The colored circles indicate genes that are up-regulated (red) or down-regulated (blue) under HERES-depleted conditions. Changes in the expression of the highlighted genes were experimentally confirmed by qRT-PCR. (B) HERES expression in the nuclear and cytoplasmic fractions of KYSE-30 cells as determined by qRT-PCR. (C) Log-scaled fold-changes of expression (x axis) and DNA methylation (y axis) of each gene in the HERES-high versus HERES-low sample groups from the TCGA dataset. The red dots indicate DE genes in ESCC. The highlighted genes are those for which there is anti-correlation between expression and DNA methylation. DEG: DE gene. N.S.: not significant. (D) The CACNA2D3 genomic locus with CpG island tracks, DNA methylation (beta value), and RNA expression (read count) in the HERES-high and HERES-low groups. (E) CACNA2D3 and LDOC1 DNA methylation patterns (methylation [M] and unmethylation [UM]) were measured by MS-PCR in KYSE-30 cells transfected with siControl or siHERES. (F) Immunoblots of histone modification markers in siControl- or siHERES-transfected KYSE-30 cells. (G) ChIP-qPCR analysis of the H3K27me3 level of CACNA2D3 in siControl- or siHERES-transfected KYSE-30 cells. Data represent the mean ± SD from 3 independent experiments. (H) Immunoblots of the products of the 3 HERES target genes (CACNA2D3, SFRP2, and CXXC4), components (β-catenin and NLK) of the Wnt/β-catenin and Wnt/Ca2+ signaling pathways, and downstream proteins associated with cell proliferation (CMYC and CCND1), invasion (MMP7), and EMT (SNAIL).
Fig. 5.
The effect of HERES on the cell cycle and apoptosis. Cell cycle (A) and apoptosis (B) assays were performed on siRNA-transfected KYSE-30 and HCE-7 cells. (A) Cell cycle analysis of siRNA-transfected KYSE-30 and HCE-7 cells by flow cytometry. The bar graph shows the percentage of cells in sub-G0, G1, S, and G2 phases in siRNA-transfected KYSE-30 and HCE-7 cell populations. (B) Apoptosis was measured by flow cytometry using PI/Annexin V staining. The bar graph represents the percentage of apoptotic cells in each population. Data represent the mean ± SD from 3 independent experiments. (C) Apoptosis markers were assessed by immunoblot in KYSE-30 and HCE-7 cells transfected with siControl or siHERES and/or pcDNA-HERES. *P ≤ 0.05, **P ≤ 0.01.
Fig. 6.
HERES regulates CACNA2D3 via interaction with EZH2. (A) Immunoblots of EZH2 and DNMT1 in KYSE-30 cells transfected with either siControl or siHERES. (B) RIP assays were performed with anti-EZH2 in KYSE-30 cell lysates. The quantity of HERES in the cell lysates (input) and the immunoprecipitates was measured by qRT-PCR. (C) IP assays were performed with anti-EZH2 in KYSE-30 cell lysates. The quantity of CACNA2D3 in the cell lysates (input) and the immunoprecipitates was measured by immunoblot. (D) Representation of the WT and mutated (HERES-Mut) HERES sequences used for IP with anti-EZH2. HERES-Mut contains a deletion of the G-rich sequence (index 1) presented in SI Appendix, Fig. S11B. (E) RIP assays were performed with anti-EZH2 in lysates of KYSE-30 cells transfected with either pcDNA-HERES or HERES-Mut (Upper). The bar graph shows the relative amount of HERES after anti-EZH2 IP using lysates of cells transfected with either pcDNA-HERES or HERES-Mut (Lower). (F) RNA FISH (40× magnification) to visualize HERES (red) and FITC staining of CACNA2D3 (green) in KYSE-30 cells transfected with pcDNA (Upper), pcDNA-HERES (Middle), or HERES-Mut (Lower). (Scale bar, 20 µm.) Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). CACNA2D3 RNA (G) and protein (H) levels were measured in KYSE-30 cells transfected with pcDNA, pcDNA-HERES, or HERES-Mut by qRT-PCR and immunoblot, respectively. Data represent the mean ± SD from 3 independent experiments (B, E, and G). **P ≤ 0.01.