Fig 2: Analysis of NUC defects in kalirin-GEF1-inhibited cells.. (A) Random walk plots and accumulated migration distances of vehicle- and kalirin–GEF1 inhibitor–treated NB-S-124 cells (13 h, 15-min intervals). Mean values + SD are presented. (B) Cell viability of NB cell lines treated with vehicle, kalirin–GEF1 inhibitor#2 (5 µM) or kalirin–GEF1 inhibitor#1 (10 µM) in 2D exclusion assay. Values are reported as mean percent + SD of vehicle-treated control. (C) Kalirin–STYV, ßIII-tubulin, and ?-tubulin co-localisation in IMR-32. (D) Violin plots showing the nucleus-to-centrosome distance in IMR-32 cells treated with kalirin–GEF1 inhibitor#1, kalirin–GEF1 inhibitor#2, or RAC1 inhibitor and in KALRN-KD or SOX11-KD IMR-32 cells. P-values: RAC1 inh: 0.0001028, kalirin–GEF1 inhibitor#1: 1.933 × 10-6, kalirin-GEF1 inhibitor#2: 1.191 × 10-5, KALRN siRNA: 0.0001527, SOX11 siRNA; 2.388 × 10-5 (Welch t test). (E) ?-tubulin distribution in control, RAC1 inhibitor–treated and kalirin–GEF1 inhibitor#2–treated IMR-32 cells. Scale bar 20 µm. (F) Representative negative images of IMR-32 in control cells and after treatment with RAC1 inhibitor, kalirin–GEF1 inhibitor#2 or after KALRN-KD stained with phalloidin-Alexa Fluor-555. Scale bar 200 µm. (G) WB for DCX in RAC1-inhibited and kalirin-GEF1-suppressed IMR-32. (H) Cell migration (16 h, 15-min intervals) in expressing DCX-RFP KALRN-KD, SOX11-KD and DCX-KD (Ambion siRNA:145587) IMR-32 cells (72 h of RNAi). Values are reported as mean + SE of control. P-values: (DCX-RFP [-] versus DCX-RFP [+]): control siRNA: n.s., DCX siRNA: 9.906 × 10-7; SOX11 siRNA: 0.1; KALRN siRNA: 3.737 × 10-7 (Welch t test). The efficiency of DCX-KD was determined by WB, and the selected siRNA is marked in red.

Fig 3: 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 4: Expression of RAC1-GEF-encoding KALRN, TIAM1, and TRIO in NB.(A) Affymetrix mRNA expression analysis (probe sets: KALRN, 230596, 206078, 227750, 232717; TIAM1, 213135; TRIO, 209011, 209012, 208178, and 209013) in two series of NB tumor samples (red) and series of various other tumor types (blue). (B) Expression of KALRN, TIAM1, and TRIO in NB cell lines (red) and series of different cancer cell lines (black). (C) Graphs showing correlation of expression of DCX (left) or SOX11 (right) and KALRN, TIAM1, or TRIO in primary NB (gse49710). (D) WB analysis of kalirin expression with kalirin-SP, TRIO, TIAM1, kalirin-STYV, and anti-kalirin-PH-2 in NB cell lines and primary tumors. Asterisks indicate previously annotated isoforms.