Cell Counting Kit-8 assay was performed as described [46 (link)]. For colony formation assay, cells were seeded in 6-well plates at a density of 1000 cells per well. After 7 days, colonies were visualized by crystal violet staining.
Serum starvation was used to induce cell cycle synchronization roughly before cell cycle distribution was analyzed by flow cytometry as described [46 (link)]. For analyzing FAM83D expression in the cell cycle, AGS cells were synchronized at G0/G1 by double-thymidine block. Briefly, cells were treated with 2mM thymidine (Sigma, USA) for 20 h, followed by culturing in fresh media for 9 h. After that, cells were treated again with thymidine for 16 h and were released from the block by washing in PBS (phosphate-buffered saline) and culturing in fresh media. Samples were taken after block at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours for flow cytometry analysis and Western blot analysis.
For the immunofluorescence analyses, tumor cells were incubated in 8-well glass slides (Millipore, USA) with primary antibodies against FAM83D (Bioss, CHINA), alpha-Tubulin (Proteintech, USA), followed by incubation with Goat anti-mouse IgG (H+L), FITC conjugate or Goat anti-rabbit IgG (H+L), TRITC conjugate (Proteintech, USA). DNA was stained with 406-diamidino-2-phenylindole.
Serum starvation was used to induce cell cycle synchronization roughly before cell cycle distribution was analyzed by flow cytometry as described [46 (link)]. For analyzing FAM83D expression in the cell cycle, AGS cells were synchronized at G0/G1 by double-thymidine block. Briefly, cells were treated with 2mM thymidine (Sigma, USA) for 20 h, followed by culturing in fresh media for 9 h. After that, cells were treated again with thymidine for 16 h and were released from the block by washing in PBS (phosphate-buffered saline) and culturing in fresh media. Samples were taken after block at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours for flow cytometry analysis and Western blot analysis.
For the immunofluorescence analyses, tumor cells were incubated in 8-well glass slides (Millipore, USA) with primary antibodies against FAM83D (Bioss, CHINA), alpha-Tubulin (Proteintech, USA), followed by incubation with Goat anti-mouse IgG (H+L), FITC conjugate or Goat anti-rabbit IgG (H+L), TRITC conjugate (Proteintech, USA). DNA was stained with 406-diamidino-2-phenylindole.
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