Fig. 4: INPP5A depletion preferentially results in elevated cytosolic calcium levels in GNAQ/11-mutant UM cells.

a, Representative images of the fluorescence signal ratio (F₃₄₀/F₃₈₀) of the ratiometric calcium indicator fura-2 in cells transduced with indicated sgRNA (mCherry⁺). Scale bar, 20 µm. b, Quantification of fura-2 fluorescence signal ratio (F₃₄₀/F₃₈₀) 72 h after transduction with the indicated sgRNAs in the depicted cell lines. Each point represents a cell (n indicates cell number quantified per group). A representative of n = 3 for 92.1 and OMM1 and n = 2 for Mel202 and Colo741 independent experiments is shown. c, Same as b for HEK293A-Cas9 parental or cells HA-GNAQ-expressing cells. A representative of n = 2 independent experiments is shown. d, Representative time-lapse images of 92.1-Cas9 cells expressing sgRNA (mCherry⁺) and calcium indicator dCys-GCaMP6 (GFP⁺). GFP is activated upon calcium binding to dCys-GCaMP6. Time is depicted in hours. Scale bar, 20 µm. e,f, Quantification of normalized GFP signal intensity of 92.1-Cas9 (e) and MeWo-Cas9 (f) cells transduced with the indicated sgRNAs. Cells were treated with the calcium ATPase inhibitor (thapsigargin 1.5 nM) at the start of imaging. A representative of n = 2 independent experiments is shown. g,h, Quantification of the percentage of dead cells of 92.1-Cas9 (g) and MeWo-Cas9 (h) cells transduced with the indicated sgRNAs and treated with the calcium ATPase inhibitor (thapsigargin 1.5 nM) at the start of imaging. A representative of n = 2 independent experiments is shown. i, Single-cell-tracking analysis of GFP intensity coupled to cell fate of 92.1-Cas9 cells transduced with sgNT (left) or sgINPP5A (right). All cells that either died or divided in randomly selected fields of view were quantified. Each row represents one single cell. A representative of n = 2 experiments is shown. All data are presented as mean ± s.e.m.; P values were determined by one-way ANOVA with multiple comparisons (b,c).

Source data