Journal: Nature Communications
Article Title: Environmental cues from neural crest derivatives act as metastatic triggers in an embryonic neuroblastoma model
doi: 10.1038/s41467-022-30237-3
Figure Lengend Snippet: a – c Kaplan–Meier analysis of overall survival probability according to RTN4R ( a ), APP ( b ) and GRIA2 ( c ) expression levels in Shi and Fischer’s published cohort (GEO: GSE62564; http://r2.amc.nl ; n = 498 samples). Raw and Bonferroni corrected p values are indicated on the graphs. d , e Representative pictures ( d ) and quantification of cell–cell cohesion rate ( e ) of IGR-N91 cell aggregates transfected with a control (siSCR) or a RTN4R siRNA (siRTN4Ra) or an APP siRNA (siAPP) or a GRIA2 siRNA (siGRIA2) and treated with E8-cSG cm ( N = 5 independent experiments; n: number of aggregates analyzed per condition; two-sided Mann–Whitney U test; ctl vs E8-cSG cm : siScr: p < 0.0001, siRTN4Ra: p = 0.0272, siAPP: p = 0.0003, siGRIA2: p = 0.0001; comparison to E8-cSG cm /siScr: E8-cSG cm /siRTN4Ra: p < 0.0001, E8-cSG cm /siAPP: p = 0.1343, E8-cSG cm /siAPP: p = 0.8601). f , g Representative pictures ( f ) and quantification of cell–cell cohesion rate ( g ) of IGR-N91 cell aggregates transfected with a control (siSCR) or a RTN4R siRNAs (siRTN4Ra, siRTN4Rb) together or not with a vector encoding for human RTN4R (pRTN4R-Myc), and treated with 10 µg/mL rOLFM1 ( N = 3 independent experiments; n: number of aggregates analyzed per condition;; two-sided Mann–Whitney U test; ctl vs rOLFM1: siScr: p < 0.0001, siRTN4Ra: p = 0.2455; rOLFM1/siRTN4Ra pCtl vs pRTN4R-Myc: p < 0.0001). h Quantification of IGR-N91 cells migration properties in transwell assays using E8-cSG cm in the lower part of the device. Cells were transfected either with a control (siSCR) or a RTN4R siRNA (siRTN4Ra) or an APP siRNA (siAPP) or a GRIA2 siRNA (siGRIA2). Ratios over the number of migrating cells in the control condition (medium without any cultured tissue in the lower part) are shown ( N = 5 independent experiments, two-sided Mann–Whitney U test; comparison to siScr: siRTN4Ra: p = 0.0079, siAPP: p = 0.4206, siGRIA2: p = 0.0159). i , j Representative pictures ( i ) and quantification of cell-cell cohesion rate ( j ) of IGR-N91 cell aggregates treated with E8-cSG cm supplemented or not with 50 μg/mL RTN4R antibody (RTN4R Ab) ( N = 3 independent experiments; n: number of aggregates analyzed per condition; using two-sided unpaired t test with Welch’s correction; ctl vs E8-cSG cm : p < 0.0001, E8-cSG cm vs E8-cSG cm + RTN4R Ab: p < 0.0001). Error bars show SEM. Scale bar: 1 mm. Source data are provided as a Source Data file.
Article Snippet: Control siRNA (siRNA scr) (siRNA Universal Negative Control #1 SIC001, Sigma-Aldrich), human RTN4Ra (NM_023004; SASI_Hs01_00129206, Sigma-Aldrich), RTN4Rb (NM_023004, SASI_Hs01_ 00229277, Sigma-Aldrich), APP (NM_000484, SASI_Hs01_00185800, Sigma-Aldrich), GRIA2 (NM_001083620, SASI_Hs01_00035395, Sigma-Aldrich). pRTN4R-Myc, human tagged ORF clone, was purchased from Origene (NM_023004; #RC204073). siRNAs were used at a concentration of 50 nM.
Techniques: Expressing, Transfection, MANN-WHITNEY, Plasmid Preparation, Migration, Cell Culture
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