human embryonic kidney derived cell lines  (ATCC)

Journal: Heliyon

Article Title: STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

doi: 10.1016/j.heliyon.2022.e12058

Figure Lengend Snippet: Over-expression of STYK1/NOK causes mitotic abnormality. (A) The endogenous STYK1/NOK expressions in different cell lines by immunoblot analysis in PC-3 cells, U-87 MG cells, DLD-1 cells, HeLa cells, HEK293T cells, RPE-1 cells and HCT116 cells. GAPDH was used as a loading control. The band intensities of STYK1/NOK were quantified with FIJI software and then normalized to that of GAPDH. The relative expressions of all others were compared to that of DLD-1 cells of which was set as 1.00. The original blots are presented in Supplementary Figure 1A and 1B. (B) Establishment of the doxycycline-controlled STYK1/NOK stable cell line. HeLa cells were infected with lentiviral constructs containing doxycycline-inducible STYK1/NOK expression cassettes. The infected cells were selected with 1 μg/mL of puromycin for 5 days to obtain the stable cell line Tet-S/N. Tet-S/N cells were treated with doxycycline for 48 h before harvesting. The original blots are presented in Supplementary Figure 1C. (C) Representative immunofluorescent images of the established stable HeLa cells that were stained with α-tubulin (Green) and DAPI (Blue). Arrows indicated cells undergoing cytokinesis. Quantification of immunofluorescent results presented in (C) by calculating proportions of mitotic cells (D) and cells undergoing cytokinesis (E) to the total cells counted. The data were mean ± SEM from four independent experiments. (F) Immunoblot analysis on doxycycline-induced STYK1 silencing. DLD-1 cells were stably transfected with lentiviral doxycycline-controlled lentiviral constructs expressing shSTYK1/NOK to establish the Tet-shS/N cell line. The transfected cells were selected with 2 μg/mL of puromycin for 5 days. Cells were cultured in the presence and absence of doxycycline for 3 days before harvesting. The original blots are presented in Supplementary Figure 1D. (G) Representative immunofluorescent images of the established stable Tet-shS/N cells that were stained with α-tubulin (Green) and DAPI (Blue). Quantification of immunofluorescent results showed the proportions of mitotic cells (H) and cytokinesis (I) as mean ± SEM from three independent experiments. Mitotic cells were distinguished and counted by morphological evaluation based on the α-tubulin and DAPI staining. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Human prostatic adenocarcinoma cell line PC-3, human brain glioblastoma cell line U-87 MG, human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line HeLa, human embryonic kidney 293 derived cell line HEK293T, hTERT-immortalized retinal pigment epithelial cell line RPE-1 and human colorectal cancer cell line HCT116 were from the American Type Culture Collection.

Techniques: Over Expression, Western Blot, Control, Software, Stable Transfection, Infection, Construct, Expressing, Staining, Transfection, Cell Culture

Journal: Heliyon

Article Title: STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

doi: 10.1016/j.heliyon.2022.e12058

Figure Lengend Snippet: CDH1 interacts with STYK1/NOK in a kinase domain dependent manner. (A) HeLa cells were co-transfected with pcDNA3.0-STYK1/NOK and FLAG-CDH1 or an empty vector. Cells were treated with 50 μg/mL of cycloheximide for the indicated time before harvesting. Immunoblot analysis (upper panel) was conducted to detect the expression of STYK1/NOK. The expression of GAPDH served as an internal control. The original blots are presented in Supplementary Figure 4A. Quantification analysis (lower panel) of the relative band intensity of STYK1/NOK was performed to determine the protein half-life of STYK1/NOK. The STYK1/NOK band intensities were normalized to that of GAPDH, then further normalized to that of t = 0 time point. (B) HEK293T cells were co-transfected with pcDNA3.0-STYK1/NOK and FLAG-CDH1 and treated with 10 μM of MG132 for 4 h before harvesting. Co-immunoprecipitation for detection of STYK1/NOK and CDH1 interaction was then performed. The original blots are presented in Supplementary Figure 4B. (C) Schematic representation of the wild type STYK1/NOK domain structure and its five D-boxes (D1 to D5). Red letters represent conserved residues and green letters represent preferred residues. (D) HEK293T cells were co-transfected with FLAG-CDH1 and pcDNA3.0-STYK1/NOK or its D-box deleted mutants (ΔD1 to ΔD5) and treated with 10 μM of MG132 for 4 h before harvesting. Coimmunoprecipitation was carried out to detect the interaction between CDH1 and either STYK1/NOK or its mutants. The original blots are presented in Supplementary Figure 4C. (E) Schematic presentation of the full-length STYK1/NOK and its truncated forms. (F) HEK293T cells were co-transfected with FLAG-CDH1 and pcDNA3.0-STYK1/NOK or its truncated forms and treated with 10 μM of MG132 for 4 h before harvesting. Coimmunoprecipitation assay was used to detect the interaction between CDH1 and full-length STYK1/NOK or its truncated forms. The star ∗ represents non-specific proteins. The original blots are presented in Supplementary Figure 4D.

Article Snippet: Human prostatic adenocarcinoma cell line PC-3, human brain glioblastoma cell line U-87 MG, human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line HeLa, human embryonic kidney 293 derived cell line HEK293T, hTERT-immortalized retinal pigment epithelial cell line RPE-1 and human colorectal cancer cell line HCT116 were from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Immunoprecipitation, Co-Immunoprecipitation Assay