Fig. 5: Identification of calcium transient inducers by MS analysis. | Nature Communications

Fig. 5: Identification of calcium transient inducers by MS analysis.

From: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins

Fig. 5

a RGS4 mRNA expression in five different BCL9 knockout RKO clones mock treated or treated with conditioned medium (CM) from wild-type RKO cells, with or without proteinase K pre-treatment. P values were calculated using Student’s t test, **P < 0.01; ****P < 0.0001; n.s., not significant. b Diagram of strategy used to identify “extracellular factor(s)” inducers of calcium waves spreading. Retention time and molecular size (M/Z) c as well as molecular structure d of small-molecules identified by MS in CM from three different BCL9 knockout RKO cell clones and compared with CM from wild-type RKO cells. e Top: heatmap of calcium transient (ΔF/F0) in the indicated ROI (red dots) in wild-type and BCL9 knockout RKO cells. White dotted line indicates the time of adding Terbutaline to the cell culture medium. Bottom: the frequency spectrum of calcium waves of indicate ROI. Red and yellow arrows arrow indicate the direction of primary and secondary way propagation, respectively. Scale bar: 20 µm. f Network of synchronous calcium transients (left, top) of calcium wave spreading and Time-lapse imaging (right) after scratching the monolayer surface in control and propranolol treated RKO cells. Left, bottom: heatmap of cell response time after scratching. Yellow: fast; blue: slow. Scale bar: 20 µm. g Left: time-lapse imaging of calcium waves in THP-1 cells co-cultured with wild-type or BCL9 knockout RKO cells. White arrows: RKO cells; Red arrows: THP-1 cells. THP-1 cells were transfected with GCAMP5 and dsRed, RKO cells were transfected with GCAMP5 only. Middle: synchronous calcium transient network of wild-type RKO and THP-1 cells co-culture system. Right: the frequency spectrum of calcium waves of wild-type and BCL9 knockout RKO and THP-1 co-culture system. Characteristic spectral lines are marked by red triangle. Scale bar: 20 µm. h RT-qPCR analysis of TGFB2 and IL10 mRNA expression in THP-1 cells treated with PMA and protein-free CM from wild-type or BCL9 knockout RKO cells. P values were calculated using Student’s t test. **P < 0.01. Data are displayed as mean ± SD. See also Supplementary Fig. 13. Source data are provided as a Source Data file.

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