Journal: Frontiers in Oncology

Article Title: ERK3/MAPK6 promotes triple-negative breast cancer progression through collective migration and EMT plasticity

doi: 10.3389/fonc.2025.1563969

Figure Lengend Snippet: ERK3 promotes collective migration and invasion of TNBC cells. (A) Transient ERK3 knockdown in the MDA-MB231 cell line by siRNA and respective scramble control (siERK3 and siWT, respectively), validated by Western blot for ERK3 and β-tubulin (βTub). (B) Transwell migration assay of siERK3 or siWT cells seeded and incubated in the top insert for 20 h under serum-free or full media conditions in the lower compartment. Nonmigrating cells were scraped from the top insert membrane, while migrating cells were detached from the bottom of the insert membrane chamber and stained for colorimetric detection. Data are presented as mean ± SD. Statistical analysis was performed by two-way ANOVA followed by Šídák’s multiple comparisons test ( N = 6). (C) Wound healing assay using siERK3 and siWT cells, measured after 20 h after the scratch; and (D) respective representative images of the wound healing assay at 0 and 20 h (scale bar: 100 µm). Data are shown as mean ± SD of the percentage relative to the shWT average wound closure. Statistical analysis was performed using a paired Student’s t -test ( N = 4). (E) MDA-MB231 siERK3 and siWT cells were seeded on different ECM substrates for 20 h, after which nonadherent cells were washed off and adherent cells were quantified using a colorimetric method (Col IV, collagen type IV; LAM, laminin; Col I, collagen type I; FN, fibronectin; TEN, tenascin; VTN, vitronectin). Data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test ( N = 5). Stable MDA-MB231 cell lines with and without shRNA-induced ERK3 silencing (shERK3 and shWT, respectively) were validated by (F) Western blot for ERK3 and GFP, with β-tubulin (βTub) detection, and by (G) qPCR for ERK3 gene expression. Gene expression was normalised to beta-actin and shown as fold change (FC), using the delta–delta CT method. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired Student’s t -test ( N = 3). (H) Wound healing assay measured after 20 h after scratch of MDA-MB231 shERK3 and shWT stable cell lines; and (I) respective representative images (scale bar: 100 µm). Data are shown as mean ± SD of the percentage relative to the shWT average wound closure. Statistical analysis was performed by unpaired Student’s t -test ( N = 4). (J) MDA-MB231 shWT and shERK3 cells were cultivated in a soft agar colony assay. After 8 days in culture, cells were solubilised, lysed, stained with CyQuant ® GR Dye, and measured by fluorometric detection. Data are presented as mean ± SD of total cell number, as determined by the standard curve. Statistical analysis was performed using an unpaired Student’s t -test ( N ≥ 6). (K) Migration and invasion ability of MDA-MB231 shWT and shERK3 spheroids at days 0 and 5 of culture in either Cultrex or collagen. Data are shown as mean ± SD of the invading area. (L) Representative images of the invasion area (white dotted lines; scale bar: 250 µm). Statistical analysis was performed by two-way ANOVA followed by Šídák’s multiple comparisons test (Cultrex N = 4; collagen I N = 6). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant.

Article Snippet: Adhesion assay to different recombinant ECM proteins was performed using the colorimetric ECM Cell Adhesion Array Kit (ECM540, Merck), plating 3 × 10 4 cells/well at 48 h post-siRNA-transfection.

Techniques: Migration, Knockdown, Control, Western Blot, Transwell Migration Assay, Incubation, Membrane, Staining, Wound Healing Assay, shRNA, Gene Expression, Stable Transfection, Colony Assay, CyQUANT Assay

Journal: Human Cell

Article Title: Establishment of Coala: a novel 3D and 2D cancer cell line derived from colorectal cancer liver metastasis

doi: 10.1007/s13577-025-01256-1

Figure Lengend Snippet: Characterization of newly-derived Coala cancer cell line. Cell line was derived from human colorectal cancer liver metastasis ( a ), forms sharply-edged, fast growing colonies in 2D ( b – d ), when embedded into Matrigel matrix, the cell line form spherically-shaped tumorspheres ( e , f ). Image g shows the phenotype of tumorspheres, when Coala cells are cultured in ultra-low attachment plates. Scale bars represent 50 µm ( b – d and k , l ) and 500 µm ( e – g ). Multicolor (M-FISH) analysis of Coala cells showing the abnormal karyotype ( h ). Negative PCR test (detection of multiple mycoplasma species) in Coala cell culture ( i ). Antibody array chip analysis shows the expression of Snail, FoxA2, E-Cadherin and Pdx-1 in Coala cell lysate. Cultured Coala cells show positivity for Cytokeratin 20 (DAPI was used for nuclei staining), scale bar represents 50 µm. Growth curve for Coala cells ( m ) and effect of 5-Fluorouracil treatment ( n ). Image ( o ) showing a tumor formed in athymic mouse 3 weeks after subcutaneous grafting of Coala cells. Images ( p ) and ( r ) show flow cytometry analysis of Lgr5 marker in early passage (P3) of Coala cells. Cells from passage No.21 were used in experiments

Article Snippet: Expression of 19 transcription factors was detected with Stem Cell Antibody Array (RD Systems, UK).

Techniques: Derivative Assay, Cell Culture, Ab Array, Expressing, Staining, Flow Cytometry, Marker