Figure 2.

Subcellular localization of LCIB-Clover in different CO₂ and pH conditions. A, Schematic of the liquid culture conditions. MBC-3 cells were cultured in medium aerated with 0.04% CO₂ at pH 7.0 for 24 h (indicated as I to the left of the white box), which was replaced with fresh medium aerated with 0.12% CO₂ at pH 7.0 (II), 0.12% CO₂ at pH 7.4 (III), 0.12% CO₂ at pH 7.9 (IV), 0.12% CO₂ at pH 8.4 (V), or 5.0% CO₂ at pH 8.4 (VI), and cultured for 3 h. Red boxes indicate the time of observation. B, Representative LCIB-Clover fluorescence images of MBC-3 cells. Roman numerals correspond to the culture conditions indicated in A. Ci concentrations in the culture medium were measured using gas chromatography, and the calculated CO₂ and ${\text{HCO}}_3^{–}$ concentrations are shown below the images. CO₂ concentrations shown in red indicate VLC (<7 µM CO₂) conditions. DIC, differential interference contrast image. Scale bars: 2 μm. C, Quantification of localization patterns of LCIB-Clover. Roman numerals correspond to the culture conditions indicated in A. The CV value of the fluorescence intensity was calculated in each cell to quantify LCIB-Clover localization. The median values derived from the analysis of MBC-3 cells are represented with error bars depicting the interquartile range (n = 20–24). Dunn’s multiple comparisons test was used to assess the statistical significance of LCIB-Clover localization between the different conditions. ^**P-value < 0.01; ^****P-value < 0.0001, Kruskal–Wallis test with Dunn’s multiple comparison.