Fig 2: GLE inhibits Lpd expression in MDA-MB-231 breast cancer cells. MDA-MB-231 (1.4 × 104) cells were grown in 5% FBS media for 24 h prior to treatment with vehicle or GLE (0.25 mg/mL, 0.96 mg/mL) for 24 h. Cells were fixed and stained with DAPI (blue) and anti-Lamellipodin (green) per manufacturer’s instructions. White arrows show lamellipodia. Experiments represent data obtained from three independent biological replicates. Zoom in each image is shown in white squares, and each share equal dimensions. Scale bars = 30 μm.

Fig 3: Lamellipodin forms a protein complex with RICTOR. (A) Number of detected proteins in unirradiated and 6 Gy-irradiated A172 cells following immunoprecipitation 1 h post-irradiation and mass spectrometry analysis (n = 2). All identified proteins are provided in Tables S4 and S5. (B) Categorization of Lpd interactome into molecular functions and pathways using Reactome and KEGG pathway classification. Comparative analysis of affected pathways upon 0 Gy and 6 Gy was done by calculation of fold change. (C) Proximity ligation assay showing interaction of Lpd and RICTOR (orange) in A172 1 h after 0 Gy or 6 Gy X-rays. Scale bar: 20 µm. (D) Quantification of proximity ligation puncta in whole cell, nucleus and cytoplasm from (C) using Fiji. Results are presented as mean ± SD (n = 3, two-sided Student’s t-test, ** p < 0.01, *** p < 0.001). (E) Representative immunoprecipitation of Lpd and non-specific control IgG. Immunoblot showing Lpd and RICTOR in copreciptates and whole-cell lysates from unirradiated and irradiated A172 cells. (F) Densitometric analyses presenting relative RICTOR binding from (E). Fold changes are calculated by normalization to Lpd and 0 Gy according to representative Western blot. Data are shown as mean ± SD (n = 3). Lpd; Lamellipodin; PLA, proximity ligation assay; IP, immunoprecipitation; WCL, whole cell lysate, IgG, immunoglobulin G.

Fig 4: Lamellipodin promotes glioblastoma cell invasion. (A) Western blot analysis of Lpd expression in whole-cell lysates of normal human astrocytes (NHA) and indicated human glioblastoma cell cultures. β-actin served as loading control. (B) Densitometry analysis of Lpd expression from Western blots in (A). Lpd intensity was measured using ImageJ and normalized to β-actin intensity. Results are represented as mean ± SD (n = 3, two-sided Student’s t-test, *** p < 0.001). (C) Representative confocal microscopy images of Lpd (green), F-actin (red) and nucleus (blue) in indicated 2D glioblastoma cells cultures plated on type I collagen. Scale bar, 20 µm. (D) Western Blot analysis of knockdown efficiency 72 h after transfection of Lpd-specific and control siRNA in indicated glioblastoma cell cultures. β-actin served as loading control. (E) Representative phase-contrast images (t = 72 h) and (F) invasion capacity of control and Lpd-depleted glioblastoma spheroids recorded 24 h, 48 h and 72 h after embedment in 3D type I collagen. Scale bar, 200 µm. Cell lines are ordered by decreasing basal invasive capacity. Results are mean ± SD (n = 3, two-sided Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001). (G) Western blot analysis of Lpd expression in whole-cell lysates from Lpd WT MEF and Lpd KO MEF. β-actin served as loading control. (H) Representative phase-contrast images (t = 72 h) and (I) invasion distance of Lpd WT MEF and Lpd KO MEF spheroids measured 24 h, 48 h and 72 h after embedment in 3D type I collagen. Scale bar: 200 µm. Results are presented as mean ± SD (n = 3, two-sided Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001). Lpd, Lamellipodin; AU, arbitrary units; si, small interfering RNA; C, control; WT, wild type; KO, knockout; MEF, mouse embryonic fibroblasts.

Fig 5: Lamellipodin promotes glioblastoma radiosensitivity via the EGFR-MAPK signaling axis. (A) Phosphoproteome analysis showing mean intensity of 55 indicated phosphorylation sites of Lpd or control depleted A172 cell cultures 1 h after 0 Gy or 6 Gy plotted as heat map (n = 4). Corresponding data are provided in Table S2. (B) Fold change in phosphorylation calculated from mean intensity from (A) normalized to 0 Gy. Alterations in phosphorylation (fold change) upon Lpd knockdown and 6 Gy compared to control knockdown are categorized as follows: increase ≥ 50% in orange, decrease ≤ 30% in blue. Corresponding data are provided in Table S3. Data are presented as mean (n = 4). (C) Identified proteins showing at least 50% increase or 30% decrease in phosphorylation site changes upon Lpd knockdown and 6 Gy X-ray in comparison to controls from (B). (D) Affected pathways with altered protein phosphorylation sites after Lpd relative to controls from (C). Enrichment of proteins in signaling pathways is indicated with p < 0.05 (Fischer’s exact test). (E) Normalized surviving fraction at 6 Gy upon knockdown of the indicated proteins subtracted by control knockdown (∆ value) showing differential effects on the surviving fraction of A172 and U343MG cells. Data were normalized to the surviving fraction at 6 Gy of the control knockdown. (F) Differences in radiosensitizing effects shown by Δ values of normalized surviving fraction 6 Gy upon knockdown of the indicated proteins in combination with Lpd knockdown subtracted by Lpd knockdown of A172 and U343MG cells. ∆ value defines altered survival compared to survival of siC (E) or siLpd#1 (F) and 6 Gy treated cells: <0 reduced survival, 0 unchanged survival, >0 increased survival. Yellow color display equal survival at 6 Gy comparable to Lamellipodin knockdown, whereas white color marks a diverging survival ∆ value. Data are represented as mean ± SD (n = 3, two-sided Student’s t-test, n.s., not significant, * p < 0.05, ** p < 0.01, *** p < 0.001). si, small interfering RNA; C, control; Lpd, Lamellipodin; SF, surviving fraction.