bt 549  (ATCC)

Journal: Clinical and Translational Medicine

Article Title: TMEM92 shields DDX3X from TTC3‐mediated degradation to confer chemoresistance in triple‐negative breast cancer

doi: 10.1002/ctm2.70681

Figure Lengend Snippet: TMEM92 stabilises DDX3X by competitively inhibiting its ubiquitination by TTC3. (A) Western blot analysis of DDX3X in control and TTC3‐knockdown (shTTC3) cells (representative blots; N = 3 independent experiments;). (B) CHX chase assay of DDX3X stability in control and shTTC3 cells (representative blots; N = 3 independent experiments). (C) In vivo ubiquitination assay showing TTC3‐mediated ubiquitination of DDX3X (denaturing IP; representative blots; N = 3 independent experiments). (D) Western blot showing rescue of DDX3X levels by shTTC3 in TMEM92‐knockdown cells (representative blots; N = 3 independent experiments). (E) In vitro GST pull‐down assay showing direct interactions between TMEM92, TTC3 and DDX3X (representative gels; N = 3 independent pull‐downs). A weak background signal was observed in the GST‐only control, consistent with low‐level non‐specific binding commonly seen in GST pull‐down assays. (F and G) Co‐immunoprecipitation (Co‐IP) assays showing the DDX3X‐dependent interaction between TMEM92 and TTC3 in MDA‐MB‐231 and BT‐549 cells. In Figure , cells transfected with Flag‐empty vector were used as a negative control for Flag‐TMEM92 overexpression (representative blots; N = 3 independent experiments). (H) In vivo competitive ubiquitination assay (denaturing IP under the indicated expression conditions; representative blots; N = 3 independent experiments). (I) In vitro binding assay showing that TMEM92 reduces TTC3 association with DDX3X (representative gels; N = 3 independent experiments). (J) Representative super‐resolution confocal images showing the spatial co‐localisation of TMEM92 (green), DDX3X (magenta) and TTC3 (yellow) in TNBC cells. Nuclei were counterstained with DAPI (blue). Scale bars = 25 µm. Enlarged views (ZOOM) highlight the overlapping signals in the perinuclear regions. Scale bars = 8 µm. Statistical analysis (for quantified datasets): two‐tailed unpaired Student's t ‐test; error bars represent mean ± SD; N values as indicated.

Article Snippet: TNBC cell lines MDA‐MB‐231 and BT‐549, and the non‐tumorigenic breast epithelial cell line MCF‐10A, were obtained from ATCC (Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Western Blot, Control, Knockdown, In Vivo, In Vitro, Pull Down Assay, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Negative Control, Over Expression, Expressing, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: TMEM92 shields DDX3X from TTC3‐mediated degradation to confer chemoresistance in triple‐negative breast cancer

doi: 10.1002/ctm2.70681

Figure Lengend Snippet: TTC3 promotes K48‐linked polyubiquitination of DDX3X. (A) In vivo ubiquitination assay using His‐tagged wild‐type (WT) and lysine‐only (K6O, K11O, K27O, K29O, K33O, K48O and K63O) ubiquitin mutants in MDA‐MB‐231 and BT‐549 cells (representative blots; N = 3 independent experiments). (B) In vivo ubiquitination assay using the His‐K48R ubiquitin mutant in MDA‐MB‐231 and BT‐549 cells to verify that TTC3‐induced polyubiquitination of DDX3X depends on K48‐linked ubiquitin chain formation (representative blots; N = 3 independent experiments).

Article Snippet: TNBC cell lines MDA‐MB‐231 and BT‐549, and the non‐tumorigenic breast epithelial cell line MCF‐10A, were obtained from ATCC (Manassas, VA, USA).

Techniques: In Vivo, Ubiquitin Proteomics, Mutagenesis