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. 2022 Apr 8;12:5944. doi: 10.1038/s41598-022-09963-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Cloning of human hnRNPD promoter and its nucleotide sequence analysis. (A) The human hnRNPD gene consists of 9 exons and 8 introns. Exons have been depicted as open boxes and numbered as E1 to E9 where as black bold lines between two boxes represents intron. The promoter region has been represented by a thin line and the forward and reverse primers used for its PCR amplification and cloning have been shown by arrows. The translation start codon (ATG) in E1 and translation stop codon (TAA) in the E8 have been marked by a vertical red line. The figure has not been drawn to scale (B) The 5ʹ-flanking region of the human hnRNPD gene along with 257 bp of first exon was PCR amplified and subjected to double stranded DNA sequencing to confirm its identity. The nucleotide sequence was analyzed using an online Transfec PROMO software (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). The putative potential transcription factors binding motifs have been shown in bold face and underlined. The primers used for generation of various promoter deletion constructs have been marked by arrows. The transcription start site mapped using RLM-RACE has been numbered as + 1 and marked with an arrow (↱). (C) The PCR amplified 5ʹ-flanking region of human hnRNPD gene was cloned upstream to the luciferase gene in pGL3-Basic and the resulting construct (pVKS-1), was co-transfected with pRL-TK plasmid in a three different oral cancer cell lines, MDA-1986 (Metastatic), SCC-4 and SCC-25 (Non-metastatic). After 48 h of transfection, the cells harvested and processed for dual luciferase assay. Renilla luciferase activity was used for normalization of transfection efficiency. Cells co transfected with pGL3-Basic and pRL-TK were processed identically and served as internal controls. Fold change in firefly luciferase activity in pVKS-1 transfected cells over the pGL3-Basic was plotted. The values are mean ± SD from three independent experiments performed in triplicate. The results were statistically analyzed using a paired two tailed Student’s t-test and values significantly different from each other have been marked by stars (**P ≤ 0.01, *P ≤ 0.05).