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TBCK Influences Cell Proliferation, Cell Size and mTOR Signaling Pathway

Liu, Yueli, Yan, Xiaoyi, Zhou, Tianhua·2013·PLoS ONE

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Figure 1 Characterization of TBCK. (A) Schematic comparison of TBCK homologs in H. sapiens, M. musculus, D. rerio, D. melanogaster, and C. elegans. Red, green and blue bars indicate possible kinase domain, TBC domain and rhodanese homology domain, respectively. (B) Expression of TBCK in mammalian cell lines. Total lysates from the indicated cells were resolved by SDS-PAGE and subjected to Western analysis. (C) Subcellular localization of TBCK. HEK293 cells grown on cover slides were immunostained with the antibodies as shown. DNA was visualized by DAPI. Bar, 10 μm.
Figure 1 Characterization of TBCK. (A) Schematic comparison of TBCK homologs in H. sapiens, M. musculus, D. rerio, D. melanogaster, and C. elegans. Red, green and blue bars indicate possible kinase domain, TBC domain and rhodanese homology domain, respectively. (B) Expression of TBCK in mammalian cell lines. Total lysates from the indicated cells were resolved by SDS-PAGE and subjected to Western analysis. (C) Subcellular localization of TBCK. HEK293 cells grown on cover slides were immunostained with the antibodies as shown. DNA was visualized by DAPI. Bar, 10 μm.CC BY · PubMed Central
Figure 2 Depletion of TBCK inhibits cell proliferation. HEK293 cells infected with the lentiviruses targeting to the various regions of TBCK mRNA (TBCK-RNAi-1 or 2) were subjected to Western blotting (A), cell counts (B), fluorescence microscopy (C) and MTT analyses (D). Bar, 200 µm. Lentivirus-infected cells were GFP-positive. Data are shown as the mean of three independent experiments ± SE (* P<0.05, ** P<0.01).
Figure 2 Depletion of TBCK inhibits cell proliferation. HEK293 cells infected with the lentiviruses targeting to the various regions of TBCK mRNA (TBCK-RNAi-1 or 2) were subjected to Western blotting (A), cell counts (B), fluorescence microscopy (C) and MTT analyses (D). Bar, 200 µm. Lentivirus-infected cells were GFP-positive. Data are shown as the mean of three independent experiments ± SE (* P<0.05, ** P<0.01).CC BY · PubMed Central
Figure 3 TBCK depletion reduces cell size. HEK293 cells were infected with the indicated lentivirus and subjected to fluorescence microscopy (A–B) and FACS analysis (C–D). (A–B) GFP-positive signals indicate the cells infected by lentivirus. Bar, 20 µm. The area measurements of infected cells were quantified by Image-Pro Plus 6.0 software. Data are shown as the mean of three independent experiments ± SE (** P<0.01, n>200). (C–D) Representative histogram of flow cytometry shows the size distribution (FSC-H) of GFP positive cells that were stained with propidium iodide. The mean of three independent experiments ± SE is shown (* P<0.05, n>100,000).
Figure 3 TBCK depletion reduces cell size. HEK293 cells were infected with the indicated lentivirus and subjected to fluorescence microscopy (A–B) and FACS analysis (C–D). (A–B) GFP-positive signals indicate the cells infected by lentivirus. Bar, 20 µm. The area measurements of infected cells were quantified by Image-Pro Plus 6.0 software. Data are shown as the mean of three independent experiments ± SE (** P<0.01, n>200). (C–D) Representative histogram of flow cytometry shows the size distribution (FSC-H) of GFP positive cells that were stained with propidium iodide. The mean of three independent experiments ± SE is shown (* P<0.05, n>100,000).CC BY · PubMed Central
Figure 4 TBCK plays a role in actin organization. HEK293 cells infected with the indicated lentivirus were subjected to immunofluorescence staining with phalloidin-TRITC (A) or anti-α-tubulin antibody (C). Lentivirus-infected cells were GFP-positive. DNA was visualized by DAPI. Bar, 10 µm. Single panel image of phalloidin-TRITC signal (B) or α-tubulin (D) is showed in rainbow palettes.
Figure 4 TBCK plays a role in actin organization. HEK293 cells infected with the indicated lentivirus were subjected to immunofluorescence staining with phalloidin-TRITC (A) or anti-α-tubulin antibody (C). Lentivirus-infected cells were GFP-positive. DNA was visualized by DAPI. Bar, 10 µm. Single panel image of phalloidin-TRITC signal (B) or α-tubulin (D) is showed in rainbow palettes.CC BY · PubMed Central
Figure 5 Depletion of TBCK influences mTOR signaling pathway. HEK293 cells were infected with the indicated lentivirus and subjected to Western blotting (A) and immunofluorescence analysis (B–C) with the antibodies as shown. GFP-positive signals indicate the cells infected by lentivirus. DNA was visualized by DAPI. Bar, 10 µm.
Figure 5 Depletion of TBCK influences mTOR signaling pathway. HEK293 cells were infected with the indicated lentivirus and subjected to Western blotting (A) and immunofluorescence analysis (B–C) with the antibodies as shown. GFP-positive signals indicate the cells infected by lentivirus. DNA was visualized by DAPI. Bar, 10 µm.CC BY · PubMed Central
Figure 6 TBCK influences the protein level of mTOR components and the activities of mTOR complexes. (A–B) HEK293 cells infected with the indicated lentivirus were subjected to immunoblotting with the antibodies as shown.
Figure 6 TBCK influences the protein level of mTOR components and the activities of mTOR complexes. (A–B) HEK293 cells infected with the indicated lentivirus were subjected to immunoblotting with the antibodies as shown.CC BY · PubMed Central
Figure 7 Depletion of TBCK represses the mRNA level of mTOR complexes components. Total RNAs from HEK293 cells infected with the indicated lentivirus were analyzed by quantitative RT-PCR with the targeted genes as shown. Error bars indicate SE (** P<0.01).
Figure 7 Depletion of TBCK represses the mRNA level of mTOR complexes components. Total RNAs from HEK293 cells infected with the indicated lentivirus were analyzed by quantitative RT-PCR with the targeted genes as shown. Error bars indicate SE (** P<0.01).CC BY · PubMed Central
Figure 8 TBCK has no significant effect on MAPK and PDK1/AKT pathway. (A–B) HEK293 cells infected with the indicated lentivirus were subjected to Western blotting with the antibodies as shown.
Figure 8 TBCK has no significant effect on MAPK and PDK1/AKT pathway. (A–B) HEK293 cells infected with the indicated lentivirus were subjected to Western blotting with the antibodies as shown.CC BY · PubMed Central

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Authors
Liu, Y., Yan, X., & Zhou, T.
Venue
PLoS ONE
Published
2013-08-19
Volume
8
Issue
8
Pages
e71349
ISSN
1932-6203
Cited by
51
References
3
Language
en

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