Fig 1: SASH1 is required for NF-κB localisation to the nucleus. (a) SASH1 is required for NF-κB stabilisation and phosphorylation. Immunoblot of SASH1-depleted cells following UVC treatment. HeLa cells were transfected with control or SASH1 small interfering RNA (siRNA) 48 h before treatment with 30 mJ/cm2 UVC. Cell extracts were isolated at the time indicated after UVC treatment, immunoblotted and incubated with the indicated antibodies. (b) Nuclear localisation of NF-κB following UVC induction with or without SASH1 depletion. HeLa cells were treated as in (a) and fixed at the indicated timepoints. Immunofluorescence was performed using NF-κB p65 antibodies. (c) Quantification and statistical analysis was performed of (b) with InCell 2200 and InCell analysis software. The data represent the average and standard deviation of three independent experiments. Unpaired T-test with *P<0.05. (d) Overexpression of SASH1 WT or 231–1247 increases NF-κB nuclear levels. Quantification of HeLa cells from fixed cells imaged with Incell 2200 and analysed with Incell analyser software. The data represent the average and standard deviation of three independent experiments. Unpaired T-test with **P<0.005
Fig 2: SASH1 overexpression induces apoptosis. (a) HeLa cells were transfected with Flag-SASH1 (OE=overexpressed) and irradiated with ultraviolet light C (UVC) 3 h to induce apoptosis (10–50 mJ/cm2). Quantification of PARP1, cleaved caspase-3 and cleaved caspase-9 levels indicated below blots wasperformed with ImageJ (University of Wisconsin, Madison, USA). Statistical analysis was performed with Student's T-test with *P<0.01 and **P<0.001. (b) Immunoblot of UVC-treated HeLa cells (3 h after 30 mJ/cm2) indicating cleavage of SASH1 from 170 to 150 kDa. PARP1, caspase-3 and caspase-9 antibodies were also used as markers of apoptosis. (c) HeLa cells were subjected to subcellular fractionation and immunoblotted. The immunoblot was incubated with SASH1, histone H3 as a chromatin marker and heat-shock protein 90 (HSP90) antibodies as a cytoplasmic marker (ThermoScientific Subcellular Fractionation Kit). (d) Representative images from immunofluorescence using SASH1 antibodies in HeLa cells following UVC (50 mJ/cm2). DAPI (4',6-diamidino-2-phenylindole) was used to stain the nucleus of the cells
Fig 3: SASH1 is cleaved by caspase-3 at D230. (a) Immunoblot of U2OS cells overexpressing N- or C-terminal Flag-tagged SASH1. (b) Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing immunoprecipitation of C-terminal Flag-tagged SASH1. (c) Schematic diagram of SASH1 protein domains indicating cleavage site and N-terminal sequencing of amino acids (GSYPTF), which are preceded by a caspase-3 recognition site (DXXD). (d) SASH1 was immunoprecipitated from SASH1-Flag stably expressing U2OS cells using M2 Flag beads, followed by induction of apoptosis by UVC (50 mJ/cm2, 3 h) with cells pre-treated (30 min) with caspase-3 inhibitor Z-VAD-FMK (20 μM). (e) SASH1 is cleaved by recombinant caspase-3. SASH1 was immunoprecipitated from U2OS cells as per (d) and incubated with recombinant caspase-3 (2 U, 16 h). (f) Ectopically expressed 230E mutant SASH1 is not cleaved following UVC exposure. HeLa cells overexpressing wild-type or D230E were treated with UVC (50 mJ/cm2, 3 h). Cell extracts were immunoblotted and incubated with the indicated antibodies. (g) SASH1 230E mutant is not cleaved by recombinant caspase-3. Cell extracts taken from HeLa cells overexpressing wild-type and 230E SASH1 were incubated with recombinant caspase-3 (2 U, 16 h)
Fig 4: Chloropyramine increases SASH1 expression in breast cancer cell lines(A–H) Cells were treated with 25 or 50 μM chloropyramine for 24 h, then lysates were prepared and SASH1 protein expression was analysed using immunoblotting. Immunoblot band intensities were quantified relative to β-actin in three independent experiments. The reproducibility and significance of changes in SASH1 expression with treatment were assessed using two-tailed t-tests. *p < 0.05, **p < 0.005.
Fig 5: SASH1 depletion partially rescues chloropyramine-induced apoptosis in breast cancer cell lines(A) Cells were transfected with negative control or SASH1 esiRNAs. After 72 h, cell lysates were prepared and SASH1 expression was analysed relative to β-actin by immunoblotting. Knockdown (KD). (B–D) Cells were transfected as above, and chloropyramine was added 24 h post-transfection. Cultures were imaged by light microscopy IncuCyte ZOOM system and digitally analysed to assess confluence relative to the untreated control at 96 h post-treatment. Data shown are means +/− the standard deviation from three independent experiments. t-tests were used to compare cell confluence with and without SASH1 depletion at each of the chloropyramine doses; *p < 0.05.
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