Figure 2
From: The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis
Cell cycle analysis of HECTD1-depleted cells. (A) Cell cycle analysis by flow cytometry PI staining in HEK293ET wild-type and HEK293ET cells treated for 48 h with either a non-targeting siRNA (NT siRNA), HECTD1 SMARTpool (SP) siRNA or the individual SMARTpool HECTD1 siRNA #6. (B) Representative images of HEK293T cells stained for EdU, phospho-Histone 3 (Ser28), Hoechst and imaged using an IN Cell Analyzer 2000 high-content microscope. Click-EdU staining was used as a readout for cells in S-phase and quantified relative to the total number of cells for: (C) HEK293T WT, KO1 and KO2, (D) HEK293T siRNA-treated, (E) hTERT-RPE siRNA-treated, or (F) NT-shRNA or HECTD1-shRNA clone 2. Data plotted as mean with error bars that represent ± S.E.M., over three experiments (n = 3 wells for each condition). Data analysed by unpaired t-test with Kruskal–Wallis. (G) Cell cycle analysis by flow cytometry PI staining. HEK293T WT or KO1 cells were synchronised in late G2 with 9 µM RO3306 for 20 h, and then released from block in full media. At each of the indicated timepoints, cells were fixed using 70% ethanol, and stained using 2 µg/ml PI, with 100 µg/ml RNase A, for 30 min at room temperature. Stained samples were then analysed immediately by flow cytometry. Gated population percentages are indicated on each graph. PI-A of 50 is equivalent to 2 N (G1 population), and PI-A of 100 is equivalent to 4 N (G2/M population). Graph showing the percentage of G1 and G2/M populations in HEK293T WT and KO1 cell lines at each time point post RO3306 release. (H) Immunoblot analysis of RIPA lysates from HEK293T wild-type, HECTD1 KO1 and KO2 cell lysates. No PARP cleavage nor or a change in p21Waf1/Cip1 levels was observed in HECTD1-depleted cells. In contrast, an increase in the levels of phospho-H3 (Ser28) was detected in both HECTD1 KO lines. GAPDH was used as loading control. To enable detection of the same samples with different antibodies, membranes were cut prior to hybridization. Uncropped western blot images are included in the “Supplementary data”, with cropped areas highlighted with a red box.
