Fig 1: HECTD1 contributes to SAC activation. (A) Experimental setup to test for the effect of HECTD1 depletion on SAC activity. (B) Immunoblot analysis showing HECTD1 levels following 48 h treatment with HECTD1 SMARTpool (SP) siRNA in HEK293ET cells. ß-actin was used as loading control. (C, D) Cell cycle analysis by flow cytometry PI staining for HEK293ET treated for 48 h with NT (Top) or HECTD1 SP siRNA (Bottom) prior to addition of DMSO (C) or Nocodazole (50 ng/ml for 18 h) (D), as shown in (A). (E) Immunoprecipitation assay of endogenous HECTD1 in HEK293T cells showing interaction with endogenous BUB3, but not MAD2 or BUBR1. Normal Rabbit IgG was used for control IP. Representative data of duplicate experiments. Note that the same results were obtained whether cells were asynchronous or arrested in mitosis through nocodazole treatment. (F) Immunoblot showing that levels of MCC components BUB3, BUBR1 and MAD2 remains similar in HEK293T WT and HECTD1 KO1 cells. (G) Immunoblot showing HECTD1 levels remain similar during S, G2 and M-phase. HEK293ET cells were synchronised in low serum for 48 h before treatment with 4 µg/ml Aphidicolin for 15 h, prior to release in complete media. Cells were harvested at the indicated time points prior to western blot analysis using anti-HECTD1, anti-Cyclin B1 (Sc-245) and anti-ß-actin. Signal intensity was quantified using ImageJ and ratios for HECTD1/ß-actin were determined. (H) HECTD1 levels remain similar during mitosis. HEK293T cells were synchronised in late G2 with RO3306 (Lane 2) or released from RO3306 block into mitosis for 10 min (Lane 3) or 30 min (Lane 4). Phospho-H3 (Ser28) was used to show the effective synchronisation using R03306, while ß-actin was used as loading control. Sample from synchronous cells is shown in Lane 1. 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.
Fig 2: HECTD1 is crucial for the functional role of LMX1B in PCa. (a) HECTD1 expression in vector and circGFRA1 transfected cells measured by qRT-PCR and western blotting using the vector group as control. (b) HECTD1 expression in circGFRA1-si-NC, circGFRA1-si-HECTD1#1, and circGFRA1-si-HECTD1#2 transfected cells measured by qRT-PCR and western blotting. (c) Cell proliferation was detected by CCK-8. (d and e) EdU assay was performed to evaluate cell proliferation. (f and g) Cell migration assessed by the transwell assay. (h) VEGF content of PCa cell supernatants. (i) IL-10 content of PCa cell supernatants. (j) TGF-β1 content of PCa cell supernatants. (k and l) The cytotoxicity of CIK cells to PC-3 and LNCap cells was determined via CCK-8 assay using the circGFRA1-si-NC group as control. Data are presented as mean ± standard error. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Fig 3: TRABID NZFs are required for trapping ubiquitin and endogenous HECTD1.A, GST pull-down assays were carried using 20 µg of GST, GST-TRABID NZF 1 to 3, or GST-TRABID NZF 1 to 3TY>LV. Loss of ubiquitin binding through TY>LV mutations abrogates binding with endogenous ubiquitin and endogenous HECTD1. B, immunoprecipitation assays showing that endogenous HECTD1, but not HERC2, binds TRABID. This data also shows that HECTD1 and HERC2 do not interact, at least in this assay. Four micrograms of either HECTD1 or HERC2 antibody was coupled to Dynabeads magnetic beads and incubated with lysates of HEK293T cells expressing either 3xFLAG-Ev or 3xFLAG-FL TRABIDC443S. Following four washes with lysate buffer, 2× LDS/100 mM DTT was added to the beads, and samples were heated for 5 min at 95 °C followed by western blot analysis. Input samples are shown on the top panel and IPs on the lower panel.
Fig 4: HECTD1 interacts with regulators of WNT signaling and glycolysis. (a) Co-immunoprecipitation (co-IP) with HECTD1 and FLAG antibodies in H9 hESCs followed by western blot against HECTD1. We loaded 13.3% of total input and 20% of total immunoprecipitated sample. The images are representative of three independent experiments. All cropped blots were run under the same experimental conditions. The original blots are included in Supplementary Fig. 13. (b) Volcano plot of the interactome of HECTD1 in H9 hESCs (n = 4). Graph represents the -log (p-value) of a two-tailed t-test plotted against the log2 ratio of protein label-free quantification (LFQ) values from co-IP experiments with HECTD1 antibody compared to control co-IP with FLAG antibody. Red colored dots beyond the curved lines indicate some of the most enriched interacting proteins after correction for multiple testing (False Discovery Rate (FDR) adjusted p-value (q-value) <0.2, s0 = 0.1). (c) Scheme indicating the Gene Ontology Molecular Function (GOMF) of HECTD1 interactors (Analysis tool: Cytoscape 3.6.0)137. (d) Bar graph representing the top GOBPs of HECTD1 interactome (P < 0.05) (Analysis tool: PANTHER138 and Gene Ontology Consortium). (e) Venn diagram represents total number and common significant interactors in hESCs, NPCs and neurons (FDR < 0.2 was considered significant, hESCs (n = 4), NPCs (n = 3) and neurons (n = 3).
Fig 5: 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.
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