Fig 2: MSI2 activated Notch1 signaling through LFNG in CD44v6+ LCSCs. a A Notch RT2 PCR Array was used to determine mRNA expression profiles between MSI2 shRNA and control CD44v6+ LCSCs. b Hypothesis diagram of MSI2 regulates Notch1 signaling pathway. c Relative mRNA of the most significantly regulated genes were detected by RT-PCR in MSI2 shRNA1 group and the corresponding control group. d Western blot showed that silencing MSI2 decreased the expression of LFNG in CD44v6+ cells while overexpression of MSI2 increased the expression of LFNG in CD44v6- cells. β-actin was used as a normalized control. e Efficiency of tumor formation of LFNG shRNA1 cells and the corresponding controls. Number of injected cells: 1 × 105. n = 4. f Representative images of spheres and histogram analysis in indicated cells. The inhibition of LFNG decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. g The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in LFNG shRNA cells compared with corresponding controls. β-actin was used as a normalized control. The inhibition of LFNG decreased the expression of stemness-related genes in CD44v6+ LCSCs. h Silencing LFNG in CD44v6+ HCC cells decreased the expression of key components of Notch1 pathway (including Notch1, NICD, Hey1 and Hes1) but MSI2 had no significant change. β-actin was used as a normalized control. i Key components of Notch1 signaling reduction caused by MSI2 knockdown could be rescued by LFNG overexpression in CD44v6+ LCSCs. For statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, t test

Fig 3: The differences expressions of LFNG, MFNG and RFNG in human tumors and healthy tissues among the 33 type cancers. (A-C). The black and yellow bar graphs indicate normal and tumour tissues of LFNG (A), MFNG (B) and RFNG (C), respectively. D-O. Pan-cancer differential expression of LFNG, MFNG and RFNG in paired tumor and adjacent normal tissues in indicated representative tumor types (LIHC, PAAD, STAD, CHOL) from TCGA database. The blue and red bar graphs indicate normal and tumor tissues of LFNG (D-G), MFNG (H-K), RFNG (L-O), respectively. (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001)

Fig 4: Analysis of drug sensitivity based on the LFNG expression in PAAD and PRAD. (A, B) Bicalutamide, lapatinib, Paclitaxel, pyrimethamine, Sorafenib, and Cytarabine, Metformin, Rapamycin, Sorafenib, Tipifarnib were relatively sensitive in PAAD and PRAD patients with higher expression of LFNG (*P < 0.05). (C, D) Camptothecin, DMOG, AP.24,534, ATRA, Vorinostat and Bleomycin, Dasatinib, CCT007093, DMOG, Z.LLNle.CHO were relatively resistant in LFNG higher expression PAAD and PRAD. (*P < 0.05)

Fig 5: The effect of LFNG on immunological status in pan-cancer. (A). Correlation between LFNG and 13 tumor-associated immune cells, as calculated using the ssGSEA algorithm. (B-G). Differences in the various immune cells proportion between groups with high and low LFNG expression in representative cancers. (ns, no significant difference. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)