Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 1. Kaplan–Meier overall survival and correlations between ERBB2 and formin mRNA expression. (A) Kaplan–Meier OS analyses of HER2-positive breast cancer samples reveal significantly poorer prognosis associated with FHOD1, INF2, and DAAM1 mRNA expression. The number of cases is denoted at the bottom of the figures. (B) ERBB2 mRNA expression in HER2-positive subtype breast tumors (n = 230) exhibits a significant positive correlation with FHOD1, INF2, and DAAM1. Kaplan–Meier analyses and correlations for all formins, excluding GRIDIP, are depicted in Supplementary Figures 1 and 2. ERBB2 = erb-b2 receptor tyrosine kinase 2; HER2 = human epidermal growth factor receptor 2; OS = overall survival; HR = hazard ratio.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Expressing

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 2. Immunohistochemistry. Tissue microarrays were sectioned at 3.5 μm and immunostained using FHOD1, INF2, or DAAM1 antibodies, following the streptavidin-peroxidase technique. The samples were evaluated based on the predominant staining intensity and categorized into four levels: negative, low, moderate, or strong. No instances of strong INF2 intensity were detected. Magnification: 400×. Scale bars = 50 µm.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Immunohistochemistry, Staining

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 3. Localization of FHOD1, INF2, and DAAM1 in MDA-MB-453 and SK-BR-3 breast cancer cell lines. Upper panel: FHOD1 (red) is predominantly seen as cytoplasmic dots. Middle panel: INF2 (red) is distributed in the cytoplasm colocalized with phalloidin (green) and lamellipodia. Lower panel: DAAM1 (red) appears as cytoplasmic dots with co-localization with actin filaments. Magnification: 400×. Scale bars = 20 µm.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques:

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 4. Small interfering RNA-mediated knockdown of FHOD1, INF2, DAAM1, or their combinations. (A) Knockdown effectiveness was assessed through immunoblotting 48-hour post-transfection, with GAPDH as a loading control. (B) Representative images of transfected cells with control, FHOD1, INF2, DAAM1, or combined siRNAs. Alexa Fluor 488-conjugated phalloidin and DAPI were used to visualize actin filaments and nuclei, respectively. Magnification: 400×. Scale bars = 50 µm. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; siRNA = small interfering RNA; DAPI = 4′,6-diamidino-2-phenylindole.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Small Interfering RNA, Knockdown, Western Blot, Transfection, Control

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 5. Effect of knockdown of FHOD1, INF2, DAAM1 or their combination in wound healing, transwell migration, and proliferation. (A) Scratch wounds were performed 48-hour post siRNA transfection, and the medium subsequently replaced by starvation medium or starvation medium supplemented with 50 ng/mL HRG to speed migration. The wound relative densities were monitored over 96 hours. (B) Forty-eight hours after transfection, cells were seeded on the upper surface of the transwell migration chamber, while the lower chamber contained medium supplemented with 10% serum as a chemoattractant. Confluence of cells on the bottom surface of the membrane was monitored for 96 h and normalized based on the initial top surface cell area for each well. (C) For proliferation assessment, cells were plated to cover 5%–10% of the area 48-hour post-transfection, and the growth of confluence measured over 144 hours. Comparisons between cells treated with control siRNA and those subjected to FHOD1, INF2, DAAM1, and their combinations were analyzed using Student’s t-test, where *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 were considered statistically significant. siRNA = small interfering RNA; HRG = heregulin.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Knockdown, Migration, Transfection, Membrane, Control, Small Interfering RNA

Journal: Journal of breast cancer

Article Title: Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

doi: 10.4048/jbc.2023.26.e47

Figure Lengend Snippet: Figure 6. HER2/ERBB2 knockdown alters FHOD1 and INF2 expression through Akt and MAPK pathways. (A) Subcellular localization of HER2 (in red) in MDA- MB-453 and SK-BR-3 cells, with ERBB2 concentrated in actin-rich cellular protrusions, observed through phalloidin staining (green). DAPI (blue) indicates nuclei. Magnification: 400×. Scale bars = 20 µm. (B) Cells were treated with ERBB2 siRNA for 48 hours, and the knockdown efficacy validated by western blotting in both MDA-MB-453 and SK-BR-3 cells. In MDA-MB-453 cells, HER2/ERBB2 knockdown decreased FHOD1, INF2, and DAAM1 levels, concomitant with a reduction in pMAPK. In SK-BR-3 cells, ERBB2 knockdown reduced FHOD1 and INF2 expression, accompanied by lowered Akt/pAkt and MAPK phosphorylation levels. Both cell lines were treated with MEK1/2 inhibitor UO126, PI3K inhibitor LY294002, or the control vehicle DMSO. Effective pathway inhibition was verified by western blotting of Akt and MAPK phosphorylation states. Treatment with PI3K or MAPK inhibitors in MDA-MB-453 cells emulated the effects of HER2/ERBB2 knockdown, decreasing FHOD1 and INF2 expression. In contrast, inhibiting PI3K or MAPK alone did not induce alterations in FHOD1 and INF2 levels in SK-BR-3 cells. Notably, DAAM1 levels remained unaffected by PI3K or MAPK inhibitor treatments in both cell lines. HER2 = human epidermal growth factor receptor 2; ERBB2 = erb-b2 receptor tyrosine kinase 2; DAPI = 4′,6-diamidino-2-phenylindole; siRNA = small interfering RNA.

Article Snippet: To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies.

Techniques: Knockdown, Expressing, Staining, Western Blot, Phospho-proteomics, Control, Inhibition, Small Interfering RNA