Fig. 2: Regulation of KCNN1 by the chimeric protein EWS::FLI1.

A KCNN1 expression (Log2 normalized expression) in A-673 cell line stably transduced with a doxycycline-inducible shRNA targeting EWS::FLI1 in control condition (−Doxycycline) or after doxycycline treatment (+Doxycycline). B. Normalized and relative EWS::FLI1 (left panel) and KCNN1 (middle panel) gene expressions in A-673/TR/shEF cell line after doxycycline treatment (1 µg/mL) (N = 5). Western-blots of SK1 and EWS::FLI1 (right panel) in A-673/TR/shEF cell line after doxycycline treatment (1 µg/mL) (N = 2). C Normalized and relative EWS::FLI1 and KCNN1 gene expressions in A-673 (N = 3), RD-ES (N = 4), and SK-ES-1 (N = 3) cell lines after 48 h of transient transfection with a pool of siRNAs targeting EWS::FLI1 (siRNA EWS::FLI1) or a siRNA control (siRNA CT) (upper panels). D Western blots of SK1 and EWS::FLI1 in A-673, RD-ES and SK-ES-1 cell lines after 72 h of transient transfection with a pool of siRNA targeting EWS::FLI1 (siRNA EWS::FLI1) or a siRNA control (siRNA CT) (N = 2) (lower panels). E Different KCNN1 transcripts according to their position on the genome. Brown rectangles correspond to transcribed and translated exons, while the salmon rectangles correspond to transcribed but not translated exons. White spaces are intronic sequences. F Normalized expression of KCNN1 transcripts (FPKM) in A-673 cell line (N = 3). G Normalized expression of KCNN1 transcripts (FPKM) in A-673/TR/SHEF cell line at day 0 and day 7 of doxycycline treatment (1 µg/mL). *: p < 0.05; **: p < 0.01 (Mann–Whitney tests). (In A, the boxes boundaries indicate the first and third quartiles - and the midline represents the median – of genes expression. The whiskers indicate the fifth and the ninety-fifth percentiles. In B, C, bars indicate means ± SD of relative and normalized EWS::FLI1 or KCNN1 mRNA expression determined by RT-qPCR. In E, F, FPKM Fragment Per Kilobase Per Million reads.).