Fig. 4: Expression of MAPK1 in BLCA patient tumor and its role in vitro and in vivo.

a The bar plot represents the expression pattern of MAPK1 across cancer types where different color gradient depict different levels of MAPK1 expression. The cancer types were abbreviated as per TCGA guidelines (https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations). b Forest plots depict the hazard ratio for pan-cancer patients, based on their MAPK1 protein expression. The brown dotted line in the middle of the plot indicates an HR of 1. The cancer types with significant p values are colored red. The size of the square boxes corresponds to the HR value. The red boxes depict having p < 0.05, while the blue boxes are non-significant. c Overall representation of a tissue microarray slide stained with MAPK1. The normal bladder tissues containing normal urothelial layer (as defined by the commercial provider) are circled in red, whereas the cancer adjacent tissues with normal urothelium (pathologically) are circled in green. The other cores (non-circled) contain muscle layers from normal tissues and are excluded from the quantitative analysis. d Enlarged representative images of a normal urothelium core sample (upper panel) and a cancer urothelium core sample (lower panel), highlighting that both cytoplasmic and nuclear staining of MAPK1 protein was observed. Scale bar = 90 µm. e Dot plots showing the H score of each cancer core and normal core in the TMA slide. The H score provides a cumulative factor undertaking both intensity and positive staining of MAPK1 in nucleus and cytoplasm individually. f Dot plot of all BLCA cell lines, curated in DepMap analysis and correlated with MAPK1 gene expression. Note the loss of MAPK1 has little negative impact on growth of some of the BLCA cell lines (blue dots) like UMUC3 and UMUC6, while loss of MAPK1 has a dramatic negative impact on growth (red dots <−0.2) in most BLCA cell lines. g mRNA and protein levels of MAPK1 gene in UMUC3 and UMUC6 cell lines in their 2D and 3D growth condition. h Graphical representation of knocking down MAPK1 in the cancer cell lines. The adjacent western blot reveals effective knockdown of the MAPK1 in the respective cell lines. i Growth curves of UMUC3 and UMUC6 in both 2D and 3D growth conditions, where the Y axis represents CyQuant fluorescence reading. j Microscopic images of spheroids obtained by mixing UMUC3_empty vector cells labeled with DiL dye (green) with UMUC3 shMAPK1 cells labeled with DiD dye (red) at a ratio of 1:1 at day 0 (top panels). The corresponding study in UMUC6 cells is shown in the bottom panels. By day 14, both UMUC6_empty vector and UMUC3_empty vector cells dominate the population. The images shown are merged images of fluorescent field and bright field. The scale bar is 3400 μm. k Growth curves of UMUC3 empty vector and UMUC3 and UMUC6 shMAPK1 xenografts in FOXN−/− mice (n = 5) show that both UMUC3 and UMUC6 shMAPK1 tumors grow significantly slower than empty vector control tumors. The inset picture shows representative tumors.