Figure 1

HECTD1 depletion reduces cell proliferation. (A, B) The Effect of transient siRNA knock down of HECTD1 on cell number was determined using trypan blue exclusion in HEK293ET (A) and HeLa (B). Data was plotted as mean with error bars that represent ± S.E.M., over three independent experiments (n = 3) defined as three separate transfections, **p < 0.01, *p < 0.05 by paired student’s t-test. (C) Immunoblot analysis showing HECTD1 transient knock down using 20 pmol SMARTpool (SP) siRNA in HEK293ET and HeLa following 72 h incubation. HEK293ET and Hela cells were transfected with lipofectamine 2000 or RNAiMAX, respectively. Cells harvested at the indicated timepoint, lysed in RIPA buffer, and lysates were analysed on a 4–12% SDS PAGE followed by western blot analysis using anti-HECTD1, and anti-β-actin antibody as loading control. (D) Cell number was also quantified in HEK293T cells and two independent HECTD1 KO cell lines, KO1 and KO2. Data was plotted as mean with error bars that represent ± S.E.M., over three independent experiments (n = 3) defined as three separate transfections, **p < 0.01, *p < 0.05 by paired student’s t-test. (E) Immunoblot analysis showing the absence of HECTD1 in HECTD1 KO1 and KO2 cell lysates. (F) Domain organisation of mouse Hectd1 highlighting HECTD1 catalytic cysteine C2579. (G) Rescue assay showing that re-expression in HEK293T KO1 of HA-FL-mHectd1 wild-type (WT), but not catalytic mutant C2579G or an empty vector control, yields a similar number of cells compared to HEK293T WT cells. Data was plotted as mean with error bars that represent ± S.E.M., over three independent experiments (n = 3) defined as three separate transfections, **p < 0.01, *p < 0.05 by a one-way ANOVA with Dunnett’s post-test. Right panel shows expression levels of HA-tagged constructs. (H) A similar trend was also observed using the Cell-Titer-Glo Luminescence cell viability assay. The indicated samples were harvested at 48 h post-transfection prior to analysis. Data plotted as mean with error bars that represent ± S.E.M., over three independent experiments (n = 3) defined as three separate transfections, *p < 0.05 by a one-way ANOVA with Dunnett’s post-test. (I) HEK293T WT cells were transfected with HA-FL-mHectd1^WT or an HA-empty vector (ev) using PEI. 48 h post-transfection cells were harvested, and cell proliferation measured (relative luminescence) with CellTiter-Glo. Data is plotted as mean with error bars that represent ± S.E.M, over three independent experiments (n = 3), **p < 0.01 by a paired student’s t-test. (J) Viable cell count (× 10⁴) for U87 cells transfected with 250 ng either HA-empty vector (Ev), HA-FL-mHectd1^WT, or HA-FL-mHectd1^C2579G(CM) using PEI. Samples were harvested at the indicated times post-transfection and counted using trypan blue to quantify the number of viable cells. Data plotted as mean with error bars that represent ± S.E.M., over three independent experiments (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05 by one-way ANOVA with a Dunnett’s post-test. Right panel shows expression levels of HA-tagged constructs in U87.