Fig 2: Kalirin is a NSC23766-sensitive RAC1-GEF in ADRN-type cells.(A) TIAM1, KALRN, and TRIO mRNA expression in MES and ADRN (marked in red color) cell lines. (B) WB analysis of TIAM1, TRIO and kalirin in a panel of primary NBs, NB cell lines. Location of antigens and RAC1-GEF domains in TIAM1, TRIO, and kalirin proteins (top) is marked by red lines and boxes, respectively. MNA status in primary tumors and cell lines is indicated. The names of ADRN cell lines are marked in red color. (C) IMR-32, SK-N-BE(2)c, and KELLY were stained with anti-STYV and anti–kalirin–SP antibodies and visualized with Cy3- or Alexa 488–conjugated secondary antibodies. Nuclei are indicated by the dashed lines. (D) WB analysis of kalirin–SP and kalirin–STYV in MNA cell lines harvested at 20% and 50% confluency. (E) RAC1 activity in IMR-32 and SK-N-BE(2)c cell treated with kalirin–GEF1 inhibitor#1 (10 μM), kalirin–GEF1 inhibitor#2 (5 μM), RHOA inhibitor (3 μM), RAC1 inhibitor (10 μM), or after SOX11 and KALRN RNAi. The cell lysates were incubated with RBD–Rhotekin or PAK-PBD beads and the bound activated RHOA and RAC1 was analysed by Western blotting. Flow through fraction was analysed for RHOA and RAC1 expression as loading control. (F) H3K4me3, H3K36me3, and H3K27me3 ChIP-seq showing binding events at KALRN promoter and gene body in NB cell lines. ADRN cell lines are marked in red color. Arrows indicate transcription start sites. (G) Expression of kalirin isoforms in primary NBs. (H) RT-PCR for 3′-most exons of kalirin-7, kalirin-12, Duet and kalirin-9 in IMR-32 and SK-N-BE(2)c. (I) KALRN gene profile obtained from RNA-seq data analysis of 27 primary NBs. 5′-most exons, 3′-most exons of the isoforms are indicated, exons encoding known and high score calpain cleavage sites (predicted for the kalirin-12 [uniprot: O60229-1]) are marked in purple color.

Fig 3: Trio is critical for Rac1 activation and invasive ability of gastric cancer cells. (A) Interaction between endogenous Filamin A with endogenous Trio in SNU484 and MKN28 cells. SNU484 and MKN28 cells were immunoprecipitated and analyzed by Western blot. The uncropped immunoblot images can be found in Figure S6. (B) Interaction between endogenous Rac1 with endogenous Trio in the presence of wild type RhoGDI2 or RhoGDI2Δ20 in SNU484 gastric cancer cells. SNU484 cells transfected with indicated constructs were immunoprecipitated and analyzed by Western blot. The uncropped immunoblot images can be found in Figure S6. (C) Interaction between endogenous Rac1 with endogenous Trio in RhoGDI2-depleted MKN28 gastric cancer cells. MKN28 cells transfected with indicated constructs were immunoprecipitated and analyzed by Western blot (left). The data are representative of three independent experiments, and relative Rac1 and Trio levels were quantified using ImageJ software (right). **, p < 0.01 as determined by t-test. The uncropped immunoblot images can be found in Figure S7. (D) Effect of Trio depletion on Rac1 activity in RhoGDI2-overexpressing SNU484 and MKN28 gastric cancer cell lines. SNU484/GDI2 and MKN28 cells transfected with indicated constructs were precipitated with a PAK1-agarose and western blotted using anti-Rac1 antibody (left). The data are representative of three independent experiments and ImageJ software was used to calculate the relative active Rac1 levels (right). For normalization, total Rac1 and α-tubulin expressions were used as controls. **, p < 0.01 as determined by t-test. The uncropped immunoblot images can be found in Figure S7. (E) Effect of Trio depletion on migration ability of RhoGDI2-overexpressing SNU484 and MKN28 gastric cancer cell lines. A wound-healing assay was used to examine the migration ability of Trio-depleted SNU484/GDI2 and MKN28 cells, with wound closure visualized using phase-contrast microscopy (left). WimScratch software (Ibidi, Munich, Germany) was used to measure the wound areas. The percentage of the wound area is expressed as the means ± SD of three separate experiments (right). ***, p < 0.001 as determined by t-test. (F) Effect of Trio depletion on invasive ability of RhoGDI2-overexpressing SNU484 and MKN28 gastric cancer cell lines. SNU484/GDI2 and MKN28 cells depleted in Trio were cultured onto matrix-coated upper chambers and the number of invading cells was measured after 24 h. The data are presented as the means ± SD of three separate experiments, carried out in triplicate. **, p < 0.01 as determined by t-test. (G) Effect of Trio depletion on proliferation rate of RhoGDI2-overexpressing SNU484 and MKN28 gastric cancer cell lines. The indicated cells were cultured at a concentration of 1 × 104 cells per well in a 6-well plate. After trypan blue staining, the viable cells were counted with a hemocytometer after 1 to 4 days of incubation.

Fig 4: Expression and localisation of kalirin, TRIO, and TIAM1 proteins in NB cells.(A) Immunolabelling of TRIO and TIAM1 in IMR-32, SK-N-BE(2)c, and KELLY grown on (collagenIV/laminin/fibronectin)-coated plastic. Scale bar 20 μm. (B) Subcellular localisation of kalirin-STYV, Golgi apparatus and γ-tubulin (left) in IMR-32 cells. Scale bar 10 μm. For superimposed images (right), centrosome-specific (centrin-2), and Golgi apparatus signals were colored green. Scale bar 5 μm. (C) Heterogeneity of kalirin-SP and kalirin-STYV subcellular localisation in IMR-32 and SK-N-BE(2)c. Scale bar 20 μm. (D) WB analysis of TIAM1, TRIO, and kalirin in IMR-32 cells after 48 h of SOX11 RNAi. (E) Migration fronts of NB-S-124 stained by kalirin-STYV and kalirin-SP. Scale bar 10 μm.

Fig 5: Modulation of KALRN levels in ADRN NB with RNAi and RA-induced differentiation.(A) Immunolabelling of kalirin in IMR-32 after KALRN RNAi. Scale bar 20 μm. (B) Western blot analysis of kalirin-8 and kalirin-STYV levels in IMR-32 and SK-N-BE(2)c after KALRN RNAi and kalirin-8 levels in IMR32 after KALRN RNAi or additional negative control siRNAs: control siRNA #2, HNRNPK siRNA, and DISC1 siRNA. (C) RAC1 activity in IMR-32 and SK-N-BE(2)c cell treated with kalirin-GEF1 inhibitor#1 (10 μM), kalirin-GEF1 inhibitor#2 (5 μM), RHOA inhibitor (3 μM), RAC1 inhibitor (10 μM), or after SOX11 and KALRN RNAi. (D) Kalrn expression in t-SNE-resolved E12.5 and E13.5 sympathetic precursors: sympathoblasts, Schwann cell precursors, bridge population and chromaffin cells (Furlan et al, 2017). (E) Sequencing electrophoregrams showing 3′-UTR of kalirin-9 isoform (top) and the exon scheme based on the results of sequencing (bottom). Location of stop codon is marked with “-.” (F) KALRN, TRIO, and TIAM expression in SK-N-BE(2)c (left) after RA (10 μM) treatment. x-axis indicate timepoints in hours. Western Blot analysis of kalirin, TRIO, and βIII-tubulin in SK-N-BE2c after 72 h of RA-treatment (top right) and of TRIO and kalirin-8 in IMR-32 and SK-N-BE(2)c after 72 h of RA treatment (bottom right). (G) Cell viability of NB cell lines treated with vehicle, kalirin-GEF1 inhibitor#2, or kalirin-GEF1 inhibitor#1. Values are reported as mean percent ± SD of vehicle-treated control.