Fig. 2

LNK promotes cellular growth and survival. a ATARiS profiles of LNK shRNA in Novartis cancer cell lines shRNA library database [https://oncologynibr.shinyapps.io/drive/]. LNK shRNA (~20 different shRNA targeting different region of LNK) were depleted/decreased in most of the melanoma cell lines (blue color) on day 14 post-transduction. b LNK promotes anchorage independent growth of melanoma cells. Left panel of each picture shows clonal growth in soft agar, while the corresponding bar graph (right panel) enumerates colony formation (mean + SD, n = 4). GFP, overexpressing GFP control; LNK OE, overexpression of LNK. *p < 0.05; **p < 0.01; unpaired t-test. c Photographs of tumor formation of murine xenografts. Upper panel, M368 xenografts grown in NSG mice show that LNK overexpressing cells formed larger tumors compared with GFP controls. Lower panel, murine melanoma B16/F10 xenograft results after Lnk silencing by CRISPR-Cas9. d LNK promotes tumorigenesis in murine xenograft model. Left and middle panels, bar graphs show tumor growth of melanoma cells overexpressing LNK (M368 and M229). Right panel, bar graphs show tumor growth of B16/F10 with silencing expression of LNK. Mean + SEM. *p < 0.05; ***p < 0.001; unpaired t-test. e, f Force expression of LNK (LNK OE) enhances cell survival and confers resistance to anoikis related cell death (induced when cells are grown anchorage-free). *p < 0.05, unpaired t-test. f Representative result of Annexin V staining after melanoma cells was grown on ultra-low binding plates. GFP, overexpression GFP control; LNK, overexpression of LNK. g Overexpression of LNK attenuates anoikis cell death. Cells were grown on ultra-low attachment plates for 48 h to induce anoikis before protein extraction. The western blot showed that overexpression of LNK reduced expression of apoptosis markers. Ctrl, control cells; LNK, cells overexpressing LNK. h LNK enhances cells survival in adverse conditions: M368 melanoma cells were grown either in nutrition deprived media (100% PBS or 1:10 diluted DMEM media, diluted with isotonic PBS) or exposed to transcription inhibitor actinomycin-D (mean + SD, n = 3). **p < 0.05, unpaired t-test. Error bars represent either SEM (d) or SD (b, e, and h). All p values were calculated using two tailed t-test