Fig 2: Lamellipodia formation in LNM35 cells. A, siCEBP?-treated LNM35 cells were used for immune cytochemistry for RAC1 by culturing in the presence or absence of ectopic 1 µmol/L C16 ceramide. Arrowheads, lamellipodia. Scale bar, 10 µm. B, siYBX1#1-treated cells were used for immune cytochemistry for RAC1 by culturing in the presence or absence of ectopic C16 ceramide. Arrowheads, lamellipodia. Scale bar, 10 µm. Quantitative results are also shown in (C) and (D) as values relative to siCTRL. Values were obtained by counting 100 or more cells and are shown as the mean ± SD. *P < .05. Experiments were repeated 3 times. E, LNM35 cells were treated with siCEBP?, then migration activities were examined with or without 1 µmol/L C16 ceramide. F, LNM35 cells were treated with siYBX1#1, then migration activities were determined with or without 1 µmol/L C16 ceramide. Experiments were repeated 3 times. G, LNM35 cells were treated with siCTRL, siCEBP?#1, siYBX1#1 or siCERS6-1, and then analyzed for the presence of the active RAC1 complex. Input and bound fractions were subjected to western blotting analyses of RAC1, PKC?, and CERS6

Fig 3: Y-box may positively regulate CERS6 expression. A, Western blot analysis results of CERS6 levels in a cancer cell line panel. CERS6 expression was quantitated as relative to ß-actin and is shown with LNCaP as a value of 1. B, Schematic illustration of insert regions of luciferase plasmids, each of which contains an insert between the indicated position and +157 promoter region. For mut1 and mut2, base substitutions in the Y- and GC-boxes are shown. TSS, transcription start site; Luc, luciferase gene; dark gray line,-112 to -88 positions; light gray line, putative p53 binding site. C-E, Luciferase activities were examined using the indicated plasmids. Data are shown as relative values to a +1 bp construct (mean ± SD) (n = 3). LNCaP and LNM35 cells were used for the analyses. *P < .05. (F) Filtering methods to screen for putative Y-box binding proteins using the GSE 11969 clinical dataset

Fig 4: Gene expression levels and invasion status. A, Correlations between gene expression and invasion status are shown. In each panel, gene expression levels in adenocarcinoma patients are shown as a waterfall plot, with each patient column associated with invasion status information shown as ++ (black) or + ~ – (white). Arrowheads indicate the threshold to classify the high (top quartile) and low (others) groups for each gene. For all genes, the expression-high groups were significantly associated with the invasive phenotype. P-values were determined using Fisher exact test. B, Representative images showing CERS6 and YBX1 expression in lung cancer specimen. AD, adenocarcinoma; NL, tumor adjacent normal tissue; SCC, squamous cell carcinoma. Scale bar, 50 µm

Fig 5: LASP1 and CERS6 modulated counterpart protein levels. (A) WB analysis of LNM35 cells was performed under knock-down conditions, as indicated. Relative values to histone H3 are shown. (B) Using LC-MS/MS, ceramide amounts were determined in siCTRL-treated (black) and siLASP1-1-treated (gray) LNM35 cells (n = 3). Quantitative values are shown as relative to siCTRL. * p < 0.05. Original western blots are presented in File S1.