Knockout of filamin A in KGN granulosa tumor cells impairs proliferation, cell cycle progression, migration, and cytoskeletal organization under mechanical stress

Figures

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  Fig. 1. FLNA expression in granulosa cell tumors (GCTs). A qPCR revealed FLNA mRNA (single band 105 bp) in GCT samples. The original gel image is shown in Suppl. Fig. S1. B Immunohistochemical staining and evaluation of FLNA expression in tissue microarrays of GCT samples. The samples exhibited expression of FLNA protein in their cytoplasm. Three representative images of different staining intensities designated as weak, moderate and strong, are shown. NC: negative control (IgG); scale bars: 50 µm

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  Fig. 2. Verification of FLNA-knockout (KO) KGN cells. A Agarose gel electrophoresis of FLNA genotyping PCR using a primer pair designed to amplify a 1031 bp fragment. Wildtype (WT) cells showed a full-length PCR product, while all four FLNA-KO clones (KO 32, KO 33, KO 35 and KO 41) exhibited a truncated product, confirming successful gene editing. Original gel image is shown in Suppl. Fig. S2A. B FLNA protein was detected in WT cells but was absent in all FLNA-KO clones. ACTB was used as a loading control. Original blot images are shown in Suppl. Fig. S2B. C Cells were stained with phalloidin-Atto 550 (green) to label actin, immunolabeled with FLNA-specific primary antibodies and Alexa-647-labeled secondary antibodies (red) to detect FLNA, and counterstained with DAPI (blue, shown in merge) to visualize nuclei. In WT cells, FLNA exhibited a filamentous staining pattern that largely colocalized with actin filaments. In contrast, FLNA staining was absent in FLNA-KO cells (only KO 32 and KO 33 are shown), confirming the loss of FLNA expression. Scale bars: 20 µm

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  Fig. 3. Proteomic analysis of FLNA-knockout (KO) KGN cells. A The Heatmap displays hierarchical clustering of differentially abundant proteins across samples. The color scale indicates protein abundance, with blue representing lower abundance and red representing higher abundance. Clustering reveals distinct patterns of protein expression between wildtype (WT) cells and FLNA-KO cells (KO 32, KO 33, KO 35 and KO 41). B The volcano plot displays differentially abundant proteins, with each point representing a protein. A total of 6,413 proteins were identified, with 979 showing significant lower abundance and 1211 showing higher abundance (q-value < 0.05, log2 fold change ≤ − 0.6 or ≥ 0.6). Proteins which were differentially abundant are highlighted in blue (downregulated) and red (upregulated). The outlier from KO 35 was excluded from the analysis. Black curves represent the permutation-based FDR significance cutoff. The ten proteins with the largest increases (red) and decreases (blue) in abundance are marked. C Overrepresentation analysis using DAVID and the “Biological Process” subset of the GO database of proteins with decreased (left panel) or increased abundance (right panel). The…

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  Fig. 4. FLNA-knockout (KO) affects morphology, and impairs proliferation and cell cycle progression of KGN cells. A Cell morphology: Fluorescence images of nuclei (upper panel) and merged phase-contrast images (lower panel) of wildtype (WT) and FLNA-KO cells (KO 32 and KO 33) reveal a change to a flattened morphology in FLNA-KO cells. Scale bar: 50 µm. B Cell size: FLNA-KO cells exhibited significantly larger cell diameters compared to WT cells. Data are presented as min-to-max box-and-whisker plots, with the box representing the interquartile range (25%–75%) and the median inside; n = 4–8 (n: number of measurements used to determine the mean diameter of trypsinized WT or FLNA-KO cells, Kruskal–Wallis test (* p < 0.05). C Cell number: FLNA-KO cells demonstrated a significantly lower proliferation rate compared to WT cells. Cell counts were measured at 24 h and 48 h post-seeding (1 × 105 cells). After 48 h, WT cells exhibited a significantly higher cell count compared to FLNA-KO cells; mean ± SEM (n = 4), two-way ANOVA (* p < 0.05). D PCNA expression: FLNA-KO cells exhibited significantly reduced mRNA expression levels of the proliferation marker PCNA, as determined by quantitative PCR…

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  Fig. 5. FLNA-knockout (KO) impairs KGN cell migration and reduce migration velocity. A Wound-healing scratch assay: Representative phase-contrast images of the wound-healing scratch assay performed with wildtype (WT) and FLNA-KO (KO 32 and KO 33) cells at 0-, 30-, and 60 h post-scratching. The percentage of confluence (C) within the wound area is indicated. Scale bar: 100 µm. Time-lapse recordings of the assay are provided in Supplementary Videos 1–3. B Wound-healing over time: The percentage of wound closure (confluence in the scratch area) was quantified for WT and FLNA-KO cells over a 0–70 h period; mean ± SEM (n = 3), two-way ANOVA (* p < 0.05). C Migration path: Spatial tracking of WT and FLNA-KO cells during the wound-healing scratch assay. All tracked cells are assumed to start from the coordinate origin. Red indicates cells migrating downward, while black represents cells migrating upward. D Directionality: Rose plots of the migration trajectories of WT and FLNA-KO cells during the wound-healing scratch assay. The plots illustrate the distribution of movement directions, with both WT and FLNA-KO cells showing a strong vertical orientation. E Directed migration velocity: The…

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  Fig. 6. Actin cytoskeletal response to laminar flow shear stress. A Actin cytoskeleton reorganization: Wildtype (WT) and FLNA-knockout (KO) cells were subjected to unidirectional laminar shear stress (25 dyn/cm2 for 24 h). As a control, cells were cultured under static conditions without flow for 24 h. Cells were stained with phalloidin-Atto 550 (red) to label actin filaments and counterstained with DAPI (blue) to visualize nuclei. WT cells reoriented their actin cytoskeleton and aligned in a vertical direction to the flow after exposure to shear stress, as indicated by the arrows showing the flow direction. In contrast, FLNA-KO cells failed to reorientate, demonstrating a lack of directional alignment in response to shear stress. Scale bars: 20 µm. B Color-coded orientation map of actin filaments: Actin filament orientations (− 90° to 90°) were visualized using color-coded maps. A more homogeneous color distribution indicates a dominant alignment direction. C Quantitative analysis of actin filament orientation: Distribution of actin filament orientations were quantified for each cell type and condition. Graphs display the frequency of actin filament orientations (y-axis) relative to…

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  Fig. 7. Transcriptional response to laminar flow shear stress. A Quantitative PCR (qPCR) revealed differential regulation of filamins and related cytoskeletal components following shear stress exposure in WT and FLNA-KO cells; mean ± SEM (n = 8), unpaired t-test (*p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001). B qPCR revealed differential regulation of genes related to adhesion-, mechanosensing and cytoskeletal remodeling following shear stress exposure after 24 h in WT and FLNA-KO cells; mean ± SEM (n = 8), unpaired t-test or Mann–Whitney test in case when no normal distribution could be assumed (*p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001)