Figure 6. Ca²⁺ transients became uncoordinated upon transiently knocking down Ano1 in organotypic cultures.

A, organotypic cultures were transduced with either non-targeting (NT) shRNA lentiviral particles or with shRNA lentiviral particles targeted to knock down Ano1 for 5 days. The relative mRNA expression of Ano1 upon transduction with non-targeting shRNA (represented as 100%) and upon transduction with shRNA-Ano1 upon normalization with β-actin (housekeeping gene). B, 3D projections of Ca²⁺ oscillations generated in organotypic cultures transduced with non-targeting shRNA lentiviral particles. Fluorescence (arbitrary units) vs. time is plotted. 2D projection of Ca²⁺ transients in lower panel shows rhythmic and coordinated Ca²⁺ transients within the ICC network. C, 3D projections of Ca²⁺ oscillations generated in organotypic cultures transduced with shRNA-Ano1. Fluorescence (arbitrary units) vs. time is plotted. 2D projection of Ca²⁺ transients in lower panel shows non-synchronous Ca²⁺ transients within the ICC network. D, measurements in Ano1 WT organotypic cultures upon shRNA-Ano1 transduction (n = 3) showed significantly less coordinated Ca²⁺ transients in ICC-MY (mean ± SEM, n = 3, *P < 0.001, unpaired t test) when compared to organotypic cultures transduced with shRNA-NT as indicated by the synchronicity index. E, frequency of Ca²⁺ transients within Ano1 WT organotypic cultures upon shRNA-Ano1 and shRNA-NT transduction were not significantly different (mean ± SEM, n = 3, P = n.s., unpaired t test).