Nstructs were identified, among which, 1516647 25 sequences appeared at least once in both experiments. The sequences and corresponding genes are shown in Table 1. This result suggest that the selection pressure was successfully applied, leading to effective enrichment of the adapted cells. However, it needs to be noted that in this approach, the selection pressure is not specific to the 
cell’s migratory capability. shRNAs promoting cell proliferation may also be enriched as they give the cells an advantage during the in vitro amplification step. Indeed, not all of the 25 genes have high percentile in the results from approach 1 (Table 1). Since approach 2 also generated pure clones harboring the 25 shRNAs, we next used these clones forsecondary screening to validate the effects of these primary hits on GBM cell migration.Validation of the screening results in vitroTwo independent cell migration assays were used to measure the migratory capability of the cell lines harboring the shRNAs we identified through RNAi screening. In the first assay, we used a Matrigel invasion chamber. After 8 hours of incubation, cells migrating to the lower surface of the membrane were stained for microscopic examination and compared to mock transduced cells produced by lentivirus harboring a scrambled shRNA sequence to determine the shRNA effect. Since U87 cells have strong migratory capability, usually thousands of cells were observed on the lower surface of the membrane. To accurately and reliably count the migrated cells, we AZ 876 chemical information developed an automated microscopic image processing program (Method S1 and Figure S1). This tool enabled us to SR-3029 web automatically quantify and statistically evaluate the results (Figure 2A and B). In the second measurement, we used 
a wound healing assay. A gap of approximately 250 mm was made by scratching with a pipette tip and the number of cells migrating across the border was monitored by time-lapse imaging. After 8 hours, cells exhibited different levels of migration until the gap was filled after 24 hours (Figure 2A). Overall, of the 25 cell lines we tested, 7 of them were observed to have significantly improved migratory capability in both assays (Figure 2A, B and C), suggesting an inhibitory role of the corresponding genes on cell motility. We mentioned above that some of the 25 primary hits didTable 1. Genes identified in the RNAi screening.Gene FIGNL1 SENP8 LCTL VAV1 HCFC1 GOLGA6L5 B3GAT2 FLNA KHSRP DLK1 PROKR1 TERF1 LRRIQ3 TWF1 NOB1 ERCC2 RIPK1 HEPHL1 SMAD1 XPO4 BUB1 AMMECR1 VPS18 DUSP12 CCNCTarget sequence CCAGGAAACAGATAGTAAT CTGGCTCAATGACCATATT GAAACTTGCTCTATCAACA GGCAGAAATACATCTACTA CAACCACCATCGGAAATAA AGCTAAACATCACCATCAT AAATAACTGCACTAAGGT CCTACTTTGAGATCTTTA CGAGAAGATTGCTCATATA CACATGCTGCGGAAGAAGA CCTGGTCCGCTACAAGAAA GTAATGATGTTGAAATGGAA CTCACTTTAACTTACCAAA CAACTTGTGATTGGATCAT CTCCTGTGCATTTAATTAA CTCACCGACTGCTTCCTGA ACCAACAGATGAATCTATA CCCAACAGGATAGGCAGTA CTATTTCATCTGTATCTT CAGCGATTCTTAAGAGTGA CAGGAAAGGTCCGAGGTTA CTCCTTCCTTCCACATTTA CATTGTACGTGCTAAATGA GTCGAAGTGTGGCCATAAT CTCCTTTCATGATAGCTTTColony frequency 68 (22.7 ) 39 (13.0 ) 33 (11.0 ) 32 (10.7 ) 20 (6.7 ) 16 (5.3 ) 12 (4.0 ) 12 (4.0 ) 11 (3.7 ) 8 (2.7 ) 6 (2.0 ) 6 (2.0 ) 5 (1.7 ) 4 (1.3 ) 3 (1.0 ) 3 (1.0 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 )Inhibition ranking 44.668.8 55.7610.1 51.8613.4 N/A 86.667.5 N/A 87.967.3 N/A 95.561.0 77.569.3 94.364.1 N/A 85.3612.8 82.968.0 90.368.8 N/A 15.8615.9 41.1614.7 85.3611.1 52.9617.3.Nstructs were identified, among which, 1516647 25 sequences appeared at least once in both experiments. The sequences and corresponding genes are shown in Table 1. This result suggest that the selection pressure was successfully applied, leading to effective enrichment of the adapted cells. However, it needs to be noted that in this approach, the selection pressure is not specific to the cell’s migratory capability. shRNAs promoting cell proliferation may also be enriched as they give the cells an advantage during the in vitro amplification step. Indeed, not all of the 25 genes have high percentile in the results from approach 1 (Table 1). Since approach 2 also generated pure clones harboring the 25 shRNAs, we next used these clones forsecondary screening to validate the effects of these primary hits on GBM cell migration.Validation of the screening results in vitroTwo independent cell migration assays were used to measure the migratory capability of the cell lines harboring the shRNAs we identified through RNAi screening. In the first assay, we used a Matrigel invasion chamber. After 8 hours of incubation, cells migrating to the lower surface of the membrane were stained for microscopic examination and compared to mock transduced cells produced by lentivirus harboring a scrambled shRNA sequence to determine the shRNA effect. Since U87 cells have strong migratory capability, usually thousands of cells were observed on the lower surface of the membrane. To accurately and reliably count the migrated cells, we developed an automated microscopic image processing program (Method S1 and Figure S1). This tool enabled us to automatically quantify and statistically evaluate the results (Figure 2A and B). In the second measurement, we used a wound healing assay. A gap of approximately 250 mm was made by scratching with a pipette tip and the number of cells migrating across the border was monitored by time-lapse imaging. After 8 hours, cells exhibited different levels of migration until the gap was filled after 24 hours (Figure 2A). Overall, of the 25 cell lines we tested, 7 of them were observed to have significantly improved migratory capability in both assays (Figure 2A, B and C), suggesting an inhibitory role of the corresponding genes on cell motility. We mentioned above that some of the 25 primary hits didTable 1. Genes identified in the RNAi screening.Gene FIGNL1 SENP8 LCTL VAV1 HCFC1 GOLGA6L5 B3GAT2 FLNA KHSRP DLK1 PROKR1 TERF1 LRRIQ3 TWF1 NOB1 ERCC2 RIPK1 HEPHL1 SMAD1 XPO4 BUB1 AMMECR1 VPS18 DUSP12 CCNCTarget sequence CCAGGAAACAGATAGTAAT CTGGCTCAATGACCATATT GAAACTTGCTCTATCAACA GGCAGAAATACATCTACTA CAACCACCATCGGAAATAA AGCTAAACATCACCATCAT AAATAACTGCACTAAGGT CCTACTTTGAGATCTTTA CGAGAAGATTGCTCATATA CACATGCTGCGGAAGAAGA CCTGGTCCGCTACAAGAAA GTAATGATGTTGAAATGGAA CTCACTTTAACTTACCAAA CAACTTGTGATTGGATCAT CTCCTGTGCATTTAATTAA CTCACCGACTGCTTCCTGA ACCAACAGATGAATCTATA CCCAACAGGATAGGCAGTA CTATTTCATCTGTATCTT CAGCGATTCTTAAGAGTGA CAGGAAAGGTCCGAGGTTA CTCCTTCCTTCCACATTTA CATTGTACGTGCTAAATGA GTCGAAGTGTGGCCATAAT CTCCTTTCATGATAGCTTTColony frequency 68 (22.7 ) 39 (13.0 ) 33 (11.0 ) 32 (10.7 ) 20 (6.7 ) 16 (5.3 ) 12 (4.0 ) 12 (4.0 ) 11 (3.7 ) 8 (2.7 ) 6 (2.0 ) 6 (2.0 ) 5 (1.7 ) 4 (1.3 ) 3 (1.0 ) 3 (1.0 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 ) 2 (0.7 )Inhibition ranking 44.668.8 55.7610.1 51.8613.4 N/A 86.667.5 N/A 87.967.3 N/A 95.561.0 77.569.3 94.364.1 N/A 85.3612.8 82.968.0 90.368.8 N/A 15.8615.9 41.1614.7 85.3611.1 52.9617.3.
