Fig 2: Lamellipodin regulates radiosensitivity and proliferation of glioblastoma cells. (A) Western Blot analysis of knockdown efficiency after transfection of Lpd-specific and control siRNA in indicated glioblastoma cell cultures. β-actin served as loading control. (B) Surviving fraction (SF) at 0 Gy relative to control knockdown and (C) clonogenic radiation survival upon 2, 4 and 6 Gy irradiation of Lamellipodin-depleted glioblastoma cell cultures after cell-line-dependent incubation times (7–15 days). (D) Western blot analysis of Lpd and β-actin expression in whole-cell lysates from Lpd WT MEF and Lpd KO MEF showing knockout efficiency. (E) Normalized surviving fraction at 0 Gy and (F) 4 Gy of Lpd KO MEF compared to Lpd WT MEF. (G) Representative Western blot analysis of transient mCherry-empty and mCherry-Lpd vector expression in U343MG. (H) Normalized surviving fraction at 0 Gy and (I) 2 Gy of mCherry-Lpd-expressing U343MG cells relative to mCherry-empty controls after 10 days. (J) Total cell number indicating proliferation of non-irradiated (circle) or 6 Gy-irradiated (triangle) A172 and U343MG cells after siRNA-mediated knockdown of Lamellipodin or control knockdown recorded at days 0 to 5. (K) Proliferation (total cell number) of non-irradiated (circle) and 4 Gy irradiated (triangle) Lpd WT MEF and Lpd KO MEF. All results show mean ± SD (n = 3–4, two-sided Student’s t-test, */# p < 0.05, **/## p < 0.01, ***/### p < 0.001). * indicates p-values of non-irradiated and # indicates p-values of irradiated conditions in (J,K). si, small interfering RNA; C, control; Lpd, Lamellipodin; SF, surviving fraction; WT, wild type; KO, knockout; MEF, mouse embryonic fibroblasts.

Fig 3: Lamellipodin and RICTOR jointly promote glioblastoma radiation survival via EGFR. (A) Normalized clonogenic radiation survival at 6 Gy of A172 and U343MG cells upon siRNA-specific triple knockdown of Lamellipodin, RICTOR and indicated proteins. Yellow columns indicate a surviving fraction comparable to double Lpd and RICTOR knockdown, whereas white columns indicate a differing survival. The pink area represents the normalized surviving fraction upon 6 Gy and Lpd and RICTOR silencing. (B) Differences in radiosensitizing effects shown by Δ values of normalized surviving fraction 6 Gy upon knockdown of the indicated proteins subtracted by double Lpd and RICTOR knockdown in A172 and U343MG cells. ∆ value is defined as survival compared to the survival of siLpd and siRICTOR and 6 Gy treated cells: <0 reduced survival, 0 equal survival, >0 increased survival. Yellow color illustrates survival at 6 Gy comparable to Lpd and RICTOR knockdown, whereas white color demonstrates a distinct survival. All results are presented as mean ± SD (n = 3, two-sided Student’s t-test, siLpd + siRICTOR/siC *** p < 0.001, siRNA/siLpd + siRICTOR * p < 0.05, ** p < 0.01, *** p < 0.001). (C) Normalized surviving fraction at 6 Gy of A172 and U343MG shown in (A). (D) Graphical scheme summarizing the interaction of Lamellipodin with RICTOR and the regulation of radiosensitivity and invasion in glioblastoma cells through interplay with the EGFR signaling hub. EGFR exerts a positive effect on Lpd phosphorylation. Conversely, Lpd negatively regulates EGFR levels and phosphorylation. This interplay is cell-line-dependent and may be modulated by basal EGFR activity. Lpd further binds to RICTOR facilitating survival and invasion. Lpd silencing dysregulates EGFR phosphorylation, renders sensitivity to X-rays and reduces invasion. si, small interfering RNA; SF, surviving fraction.

Fig 4: Lamellipodin and RICTOR jointly regulate invasion and radiosensitivity in glioblastoma cells. (A) Representative Western blot and (B) normalized surviving fraction of non-irradiated and 6 Gy irradiated A172 cells upon Lpd and RICTOR knockdown. (C) Representative images of spheroids in 3D collagen I matrix 72 h after seeding. (D) Invasion at 24 h, 48 h and 72 h of control and RICTOR siRNA treated A172 and U343MG. (E) Differences in radiosensitizing effects shown by Δ values of normalized surviving fraction 6 Gy upon knockdown of the indicated proteins subtracted by RICTOR knockdown in A172 and U343MG cells. ∆ value describes an altered survival relative to the survival of siRICTOR and 6 Gy treated cells: <0 reduced survival, 0 equal survival, >0 increased survival. Yellow color displays similar survival at 6 Gy comparable to RICTOR knockdown, whereas white color mark a differing survival. (F) Western blots of Lpd, RICTOR, phospho-RICTOR T1135 and β-actin in Lpd silenced A172 and U343MG cells 1 h after 0 Gy and 6 Gy irradiation. (G) Densitometry of RICTOR and phosphorylated RICTOR T1135 from (F). Fold changes of RICTOR levels are calculated by normalization to β-actin. Fold changes of phosphorylated RICTOR are determined by normalization to total RICTOR expression. All protein levels are related to the corresponding β-actin. Fold changes are relative to control-treated and unirradiated samples. (H) Representative immunoblot showing RICTOR and RICTOR T1135 upon EGFR knockdown and 1 h after irradiation in A172 and U343MG. Actin served as loading control. (I) Fold changes of total RICTOR and phosphorylated RICTOR T1335 calculated from (H). Fold changes of RICTOR levels are calculated by normalization to β-actin. Fold changes of phosphorylated RICTOR are determined by normalization to total RICTOR expression. All protein levels are related to the corresponding β-actin. Fold changes are relative to control treated and unirradiated samples. All results are presented as mean ± SD (n = 3–4, 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.

Fig 5: 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.