Fig. 4

TNBC are more sensitive to GBP1 knock-down than non-TNBC cells. EGFR drives GBP1 expression. a GBP1 mRNA levels were evaluated via quantitative PCR in different cell lines. b GBP1 knock-down (shGBP1) using pLKO.mKO2 for 96 h affected the growth of TNBC cells more effectively than that of non-TNBC cells, as assessed using two shRNA sequences. An shRNA targeting non-human gene luciferase (shLuc) was used as a control. Data were split between cells that died (left) and cells that proliferated less (right) after knock down. c Representative fluorescence microscopy images of MDA-MB-231 after 96 h of GBP1 knock-down compared with shLuc. DAPI staining of nuclei is shown in blue, and mKO2 fluorescence of cells positive for viral transduction is shown in yellow. d Cell proliferation assay (performed over 7 days) of cell lines selected to stably express the shGBP1 and shLuc sequences. e Propidum iodide incorporation assay was performed to evaluate the fraction of cells that are in apoptosis/late necrosis state. EGFR is more highly expressed in TNBC than non-TNBC tissues (f, top) and cell lines (f, down). The FDR value was absent in DESeq2 comparisons due to outlier removal. g GBP1 and EGFR expression levels are highly correlated in tissues (left) and cell lines (right). h GBP1 expression level positively correlates with EGFR total protein level. Log₂-transformed upper-quantile RSEM expression values were used, with whiskers extending to half of the interquartile range. Gray circles denote each sample Notches denote the 95% confidence interval of the median. (I) MDA-MB-231 cells were serum starved for 24 h and then stimulated with 50 ng/mL of EGF for six hours. Western blotting (right) confirmed that the treatment increased EGFR stimulation (increase of Tyr1068 phosphorylation). qPCR (left) showed that, with the exception of BT549, all tested cell lines responded to EGF stimulation by increasing GBP1 expression. Error bars denote one standard error of the experimental triplicates