Journal: Cancer Research Communications

Article Title: MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases

doi: 10.1158/2767-9764.CRC-23-0090

Figure Lengend Snippet: MARCH2 promotes ubiquitination and downregulation of SNAIL. A, HA-tagged WT, but not RING domain–mutant (W97A), MARCH2 downregulates SNAIL levels in MDA-MB-231 cells. B, MARCH2 coprecipitates with SNAIL in MDA-MB-231 cells coexpressing FLAG-SNAIL and HA-MARCH2. C, WT, but not RING domain– or transmembrane domain–mutant, MARCH2 ubiquitinates SNAIL. FLAG-Snail was immunoprecipitated from MDA-MB-231 cells coexpressing FLAG-Snail, HA-Ubiquitin and GST-tagged MARCH2 constructs. Immunoprecipitates were blotted with antibodies to HA to assess ubiquitination. D, MARCH2 ubiquitinates SNAIL in in vitro ubiquitination assay. Reactions were performed with recombinant SNAIL (Myc-tagged) in the presence of ubiquitin, recombinant E1 (UBE1), recombinant E2 (UBE2D3) and/or recombinant MARCH2.

Article Snippet: The in vitro ubiquitination reactions were performed using 2 µmol/L recombinant human SNAIL protein (Origene TP304581), 3 µmol/L recombinant human March2 E3 ligase (Origene TP307517), 100 µmol/L Ubiquitin (R&D Systems U-100H-10M), 100 nmol/L recombinant E1(UBE1, Biotechne E-305), 500 nmol/L recombinant E2 (UBE2D3, Biotechne E2-627), 1X ATP Energy Regeneration buffer (Enzo BML-EW9810-0100) in a 25 µL reaction buffer containing 50 mmol/L Hepes, pH 8.0, 50 mmol/L NaCl, and 1 mmol/L TCEP.

Techniques: Mutagenesis, Immunoprecipitation, Construct, In Vitro, Ubiquitin Assay, Recombinant

Journal: Biomedical Journal

Article Title: High expression of embryonic stem cell marker SSEA3 confers poor prognosis and promotes epithelial mesenchymal transition in hepatocellular carcinoma

doi: 10.1016/j.bj.2023.100612

Figure Lengend Snippet: SSEA3Cer-enhanced hepatocellular carcinoma (HCC) cancer cell migration and invasion were inhibited by ZEB1 silencing. (A) Effect of SSEA3Cer on expression level of transcription factors regulating migration and invasion related genes. HA22T, HA59T, and Hep3B cells were incubated with glucosylceramide (GlcCer) (Ctrl, left panel), 30 μM SSEA3Cer (middle panel) for 24 h. Total RNA was extracted for quantitation of SNAIL, TWIST1, and ZEB1 by quantitative real-time PCR. The mRNA levels were normalized to the level of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and compared to the control cells. (B) HA22T, HA59T, and Hep3B cells were incubated with GlcCer (Ctrl, left panel), 30 μM SSEA3Cer (middle panel) or SSEA3Cer (30 μM)/SSEA3 Ab (5 μg) (right panel) for 24 h. Total RNA was extracted for quantitation of ZEB1 by quantitative real-time PCR. The mRNA levels were normalized to the level of GAPDH and compared to the control cells. (C–E) Boyden chamber assay was employed to examine the migration and invasion ability of HA22T, HA59T, and Hep3B cells. The cells were transfected with control siRNA (si-Ctrl) or ZEB1 siRNA (si-ZEB1) for 24 h. Then, the migration (C), invasion (D), and quantitative real-time PCR (E) assays were performed in the cells incubated with GlcCer or 30 μM SSEA3Cer for 24 h. Total RNA was extracted for quantitation of CDH1, CDH2, VIM, FN, MMP2, and ZEB1 by quantitative real-time PCR. The mRNA levels were normalized to the level of GAPDH and compared to the siCtrl/GlcCer cells. The data were presented as mean ± SD of triplicate determination. The results were from three independent experiments ( ∗ p < 0.05, One-way ANOVA test).

Article Snippet: Primer pairs for CDH1 (HP207683), CDH2 (HP205580), VIM (HP206907), FN (HP234005), MMP2 (HP207826), SNAIL (SNAI1, HP209016), TWIST1 (HP200446), ZEB1 (HP215380) and GAPDH (HP205798) were purchased from OriGene (Rockville, MD, USA).

Techniques: Migration, Expressing, Incubation, Quantitation Assay, Real-time Polymerase Chain Reaction, Control, Boyden Chamber Assay, Transfection