Journal: Materials Today Bio

Article Title: Cellular EMT-status governs contact guidance in an electrospun TACS-mimicking in vitro model

doi: 10.1016/j.mtbio.2024.101401

Figure Lengend Snippet: (a) Relative expression of CDH1 and VIM mRNA in MCF7_miR200c_KO and DOX-treated MDA-MB-231_i-miR200c (MB231_i-miR200c) cells normalized to wild type (unmodified MCF7 or untreated MB231_i-miR220c, respectively). (b) Western blot of MB231_i-miR200c cells with and without doxycycline-induction stained for EMT-relevant marker CDH1, VIM and CD44s. (c) Whiskers plot showing alterations in C N -values (from 40× images) in MB231_i-miR200c cells with and without doxycycline-induction for growth on TACS5- and TACS6-like structures. One-way ANOVA with Bonferroni multiple comparison test was performed to calculate P-values at a 95 % confidence interval (n (cells) > 50). (d) Corresponding confocal images of (c) at 10× magnification. Cells were stained for nuclei (blue) and the cytoskeleton (red). Comparison of trajectory analysis of (e) WT cells with MCF7_KO or (f) MB231_i-miR200c, respectively, performed with Fiji software (TrackMate). Displacement, mean migration speed, confinement ration and mean directional change were plotted as whiskers plot (5–95 percentiles). Statistical significance was assessed with a t -test based on > 150 cells/condition. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05, ns = not significant).

Article Snippet: Human CD44s Pan Specific Antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Staining, Marker, Comparison, Software, Migration

Journal: Oncogenesis

Article Title: SALL4 promotes gastric cancer progression through activating CD44 expression

doi: 10.1038/oncsis.2016.69

Figure Lengend Snippet: SALL4 knockdown reduces the expression of stemness and EMT-related genes and the activation of ERK, STAT3 and NF-κB pathways. ( a ) Quantitative RT-PCR analyses of Oct4, Sox2, Nanog, c-Myc and CD44 expression in control and SALL4 knockdown gastric cancer cells. ( b ) Western blot analyses of Oct4, Sox2, Nanog, c-Myc and CD44 expression in control and SALL4 knockdown gastric cancer cells. ( c ) Western blot analyses of p-ERK, ERK, p-p65, p65, p-STAT3 and STAT3 expression in control and SALL4 knockdown gastric cancer cells. ( d ) Quantitative RT-PCR analyses of CD44s and CD44v6 expression in control and SALL4 knockdown gastric cancer cells. ( e ) Western blot analyses of CD44s and CD44v6 expression in control and SALL4 knockdown gastric cancer cells. ( f ) CD44s and CD44v6 expression in tumour tissues and non-tumour tissues was detected in a tissue array by using immunohistochemistry. ( g ) The patients were divided into high or low group based on CD44s and CD44v6 expression. The overall survival probability was assessed by using the Kaplan–Meier curve. * P <0.05, ** P <0.01, compared to sh-Ctrl group.

Article Snippet: Tissue array was incubated with antibodies against CD44s (MAB7045; R&D Systems, Minneapolis, MN, USA) and CD44v6 (AB2080; Millipore, Billerica, MS, USA).

Techniques: Knockdown, Expressing, Activation Assay, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry

Journal: Journal of Cancer

Article Title: TGF-β2: A Novel Target of CD44-Promoted Breast Cancer Invasion

doi: 10.7150/jca.6638

Figure Lengend Snippet: Time-course western-blot validation of the tetracycline (tet) regulated CD44 expression in MCF7F-B5 breast cancer cell line. Protein lysates were collected at different time points (18, 24 and 48hrs) following withdrawal of tet, in the presence of hyaluronan (HA). The level of expression of CD44s showed a time-dependent increase and was maintained up to 48 hrs.

Article Snippet: The membrane was probed with mouse anti-human CD44s monoclonal antibody (1:500 dilution; R&D Systems,) and then treated with a sheep anti-mouse IgG/horseradish peroxidase conjugate (1:2000 dilution; Santa Cruz Biotechnology).

Techniques: Western Blot, Biomarker Discovery, Expressing

Journal: Oncogene

Article Title: Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

doi: 10.1038/sj.onc.1209628

Figure Lengend Snippet: Figure 1 Validation of inducible tetracycline-regulated CD44 expression in MCF7F-B5 cells. (a) Northern blot demonstrating time-dependent induction of CD44 mRNA in MCF7F-B5 cells following the removal of tet from the culture media. Equivalent loading of RNA samples was confirmed by detection of 18S mRNA. (b) Immunoblot demonstrating time-dependent induction of CD44s (85 kDa band) protein expression in MCF7F-B5 cells following tet withdrawal. (c) Immunocytochemistry demonstrating punctate cell-surface distribution of CD44 on MCF7F-B5 cells when cultured in the absence of tet (top panel). Induction of CD44 expression was concomitant with increased cell surface binding of FITC-conjugated HA (bottom panel) detected following the removal of tet from the growth media.

Article Snippet: Cells were washed in PBS and incubated with a 1:1000 dilution of clone 2C5 mouse anti-human CD44 antibody (R&D Systems, Abingdon, UK) for 30min at room temperature.

Techniques: Biomarker Discovery, Expressing, Northern Blot, Western Blot, Immunocytochemistry, Cell Culture, Binding Assay

Journal: Oncogene

Article Title: Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

doi: 10.1038/sj.onc.1209628

Figure Lengend Snippet: Figure 2 Functional validation of inducible tetracycline-regulated CD44 expression in MCF7F-B5 cells. (a) tet-regulated induction of CD44 expression demonstrated by immunoblotting coincides with increased (b) migration and (c) invasion of MCF7F-B5 cells (left panel), determined by in vitro Boyden chamber assays. All data points shown are the mean7s.e.m. of three independent experi- ments, each conducted in triplicate. Statistically significant differences between data points were determined using a Student’s two-tailed t-test. *Po0.05; **Po0.01, ***Po0.001. (c) Right panel: Representative images of the Boyden chamber membrane captured under low magnification are shown to illustrate the difference in cell invasion potential upon the induction of CD44 in MCF7F-B5 cells.

Article Snippet: Cells were washed in PBS and incubated with a 1:1000 dilution of clone 2C5 mouse anti-human CD44 antibody (R&D Systems, Abingdon, UK) for 30min at room temperature.

Techniques: Functional Assay, Biomarker Discovery, Expressing, Western Blot, Migration, In Vitro, Two Tailed Test, Membrane

Journal: Oncogene

Article Title: Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

doi: 10.1038/sj.onc.1209628

Figure Lengend Snippet: Figure 4 Time-dependent regulation of cortactin expression by HA/CD44 signaling in MCF7F-B5 cells. (a) Results of qPCR conducted on RNA isolates extracted from MCF7F-B5 cells over a 12 h time course following stimulation with HA (100 mg/ml). Cells were cultured in tet-free media for 24 h before experimentation to ensure induction of CD44 expression. All values were normalized against 18S mRNA expression and fold-changes calculated by comparison of cortactin mRNA levels determined in the absence of HA stimulation. (b) Immunoblot confirming time-dependent increase in cortactin protein upon HA-stimulation (100 mg/ml) of MCF7F-B5 cells cultured in the absence of tet. Equal protein loading was confirmed by re-probing the membrane for b-tubulin expression.

Article Snippet: Cells were washed in PBS and incubated with a 1:1000 dilution of clone 2C5 mouse anti-human CD44 antibody (R&D Systems, Abingdon, UK) for 30min at room temperature.

Techniques: Expressing, Cell Culture, Comparison, Western Blot, Membrane