Fig 2: CRISPR-cas9 mediated deletion of genomic locus of Sema4B results in reduced clonal potential. (A,B) Colony formation assay experiments (n = 3) were performed for cell line mutated with single CRISPR ((A), clone 1, 2 and CRISPR cont 1) or cells with CRISPR assisted deletion of the genomic locus of Sema4B ((B), del 1, del 2 or CRISPR control 2). 400 cells were seeded in each 6-well plate and stained with Giemsa after 18 days. Colonies were counted for each well; data represent the median of 9 plates in three experiments (the p values were calculated with the one-tailed Wilcoxon-Mann-Whitney Test * = 0.0328, *** = 0.0567668E-5).

Fig 3: Expression of Sema4B in glioma cell lines. (A) Representative image of a western blot analysis of human glioma cell line extracts reveals expression of Sema4B in all cell lines tested. Actin was used as a loading control. The full-length blots are presented in Supplementary Fig. 3. (B) qPCR analysis of Sema4B mRNA in different glioma cell lines. Data are presented as mean ± s.e.m. (C) qPCR analysis of Sema4B mRNA under conditions of hypoxia. U87-MG and A127 were exposed to 1% or 21% oxygen for 6 h. Data are presented as mean ± s.e.m. The results in (A–C) are representative of three independent repetitions.

Fig 4: CRISPR-cas9 mediated mutations and deletion of almost the entire genomic locus of Sema4B do not effect proliferation. (A) Map of the genomic locus of Sema4B showing the potential alternative splicing, location of the gRNA targets and PCR targets used for the analysis of the genomic locus after CRISPR-cas9 mediated mutations or deletion. (B) Representative western blot of U87-GM cell pools treated with sh-cont1 or shRNA1 or 2. In addition protein extracted from cell clones generated by CRISPR-cas9 and cont1 gRNA (clone1), gRNA1 (clone 1) or gRNA 2 (clone 2) targeting the signal sequence of Sema4B are shown. The full-length blots are presented in Supplementary Fig. 4. (C) qPCR analysis of the same CRISPR cell clones demonstrate a variable effect on mRNA expression of individual clones. (D) Resazurin cell viability assay was used to evaluate the CRISPR clones in cell proliferation assay. Cells were tested one day after plating and four days in culture. The fluorescent read on day 4 was normalized to the read of the same cell line on day 1. No effects on proliferation are detected. (E) Representative western blot of U87-GM cell clones generated by two rounds of CRISPR-cas9 with two sets of gRNA in each round (the full-length blots are presented in Supplementary Fig. 4). One control line (cont2) and two deletion lines are shown (del1 and del2). Two cell clones were negative for oligo sets c,d and e,f (both were positive for fragment a,b) qPCR analysis of the same del1 and del2 cell clones show that mRNA levels of both clones are very low with del 2 almost undetectable. (G) Resazurin cell viability assay was used to evaluate del1 and del2 clones in cell proliferation assay. Cells were tested one day after plating and four days in culture. The number of cells on day 4 was normalized to the same cell line on day 1. No effects on proliferation are detected.

Fig 5: Knockdown of Sema4B inhibits proliferation and induces cell death. (A) qPCR results show that knockdown of endogenous Sema4B by three different shRNA sequences reduces the expression of this gene in U87-MG cells (data are presented as mean ± s.e.m., Mann–Whitney U test, n = 3). (B) XTT assay was used to evaluate U87-GM cell number at 1, 2, 3 and 4 days after plating. Each group of cells was treated with lentivirus expressing shRNA targeting Sema4B or control. The cells were plated for XTT assay 48 h after infection (data are presented as medians with range). P values were calculated with one-tailed Wilcoxon Signed Ranks Test (n = 5). (C–H) Effect of sh-cont and two sh-Sema4B were tested on U87-MG (C,E,G) or G55TL (D,F,H). (C,D) The same fields were monitored from 48–168 h (C) or 48–96 h (D) after infection (data are presented as mean ± s.e.m., n = 3). The p values were calculated with the Chi-square Fisher’s Exact test. (G,H) Glioma cell death was monitored using live/dead assay from 48 h up to 168 h (E) or up to 96 h (F). Data represent the means ± s.e.m. of three experiments (n = 3); p values were calculated with Chi-square Fisher’s Exact test. (G,H) Cell proliferation was monitored by adding a BrdU pulse 2 h before fixation. Proliferation was tested at 24–72 h (G) or 48–96 h (H). Percentage of BrdU-positive cells of the total DAPI-positive cells in each field is presented. Data represent the means ± s.e.m. of three experiments (n = 3); p values were calculated with Chi-square Fisher’s Exact test. *P < 0.05, **p < 0.001 ***p < 0.001.