Fig. 4.

LCK and LYN phosphorylation and EMT are reduced by NDBT knockdown

(A-B) Western blot analysis was utilised to validate the phospho-kinase arrays and investigate the expression of LCK and LYN signalling effectors in response to hypoxia and NDBT knockdown. Hypoxia increased phosphorylation of tyrosine kinases LCK and LYN, whereas NDBT knockdown reduces phosphorylation of LCK and LYN.

(C-F) Wound healing assays were used to identify that LCK and LYN inhibition reduces migration in four triple negative breast cancer cell lines in normoxia and hypoxia (1% O₂). (G-J) Invasion through a matrigel coated boyden chambers was reduced by LCK and LYN inhibition in normoxia and hypoxia in four triple negative cancer cell lines.

(K-R) QRT-PCR analysis was used to identify that EMT transcription factors (Twist, ZEB1, Snail and Goosecoid) gene expressions are reduced by NDBT knockdown in normoxia and hypoxia. (S) NDBT knockdown reduces mesenchymal markers Vimentin and ZEB1 expression and increases epithelial marker E-cadherin expression in HCC1806. (T) NDBT knockdown reduces mesenchymal marker ZEB1 expression in MDA-MB-231. shCTL=control shRNA cells, sh4A4/sh4A5= SLC4A4/SLC4A5 targeting shRNA knockdown cells where multiple shRNAs were used to target the same gene this is denoted by -1, -2, -3, S0859=NDBT inhibitor (ANOVA/t-test as appropriate, ***p<0.001, **p<0.01, *p<0.05, significant relative to shCTL, n=3).