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. 2019 Oct 10;9:14581. doi: 10.1038/s41598-019-51056-5

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Establishment of the doxycycline-regulated H9c2-CK2α-44 cell line with inducible silencing of CK2α. (a) H9c-2 cells were transduced with lentiviral particles carrying a SMARTchoice inducible CK2α-shRNA construct containing a turbo-GFP (tGFP) reporter gene (upper). Expression of CK2α-shRNA is induced in the presence of doxycycline (lower). (b) The H9c-2-derived cell line (i.e. H9c2-CK2α-44) stably incorporating the construct was analyzed by flow cytometry in the presence of 1 µg/ml doxycycline for up to six days. Quantification of green fluorescence emission (tGFP-positive cells) indicative of the efficiency of shRNA transcription is shown in the graph. Dashed line indicates the median levels of tGFP expression after two days of incubation for both doxycycline-treated (grey peak) and control cells (black peak). Fluorescence-based pictures of cells showing increasing expression of tGFP in the presence of doxycycline are shown on the right side. Cell nuclei were visualized by Hoechst 33258 staining. (c) Median fluorescence is shown in the dot plot in arbitrary units. Vehicle control (CT) at six days is depicted. Two independent experiments were carried out. Results from one representative experiment are shown. (d) H9c2-CK2α-44 cells were treated with vehicle (i.e. dd water) or with 1 µg/ml doxycycline (Dox) for increasing amounts of time. Whole cell lysates were analyzed by Western blot employing a mouse monoclonal antibody against CK2α and CK2α’. β-actin detection served as loading control. (e) H9c2-CK2α-44 cells and the parental cell line were harvested after 0, 2 and 3 days of treatment with vehicle (−) or 1 µg/ml doxycycline (+) and whole cell lysates were analyzed by Western blot employing the indicated antibodies. β-actin detection served as loading control. (f) H9c2-CK2α-44 cells were incubated with 1 µg/ml doxycycline for three days, transfected with scr-siRNA and CK2α’-siRNA for three days, respectively, as indicated in the figure. Last lane refers to cells treated with doxycycline and transfected with CK2α’-siRNA for three days. Whole cell lysates were analyzed by Western blot employing the indicated antibodies. All experiments were performed three times obtaining similar results; one Western blot experiment of three is shown. Abbreviations: LTR: 5’ Long Terminal Repeat; Ψ: Psi packaging sequence; RRE: Rev response element; P_TRE3G: Inducible promoter with tetracycline response elements; P_mCMV: SMARTchoice promoter; Puro^R: Puromycin resistance; 2a: Self-cleaving peptide; Tet-On 3G: Doxycycline-regulated transactivator protein; WPRE: Woodchuck hepatitis post-transcriptional regulatory element; 3’SIN LTR: 3’ Self-inactivating long terminal repeat.