Figure 6.

The requirement of LCIC for the LCIB-Clover migration and relocation. A, Schematic of the liquid culture conditions. C16-B cells were cultured in medium aerated with 5% CO₂ illuminated at 120 µmol photons m⁻² s⁻¹ (white box) for 24 h, which was replaced with the fresh medium aerated with 0.001% CO₂ for 6 h (indicated as I to the left of the white box). C16-BC cells were cultured in medium aerated with 5% CO₂ for 24 h (II), which was replaced with the fresh medium aerated with 0.001% CO₂ for 3 h (III) or 6 h (IV) in the light or for 3 h (VI) or 6  h (VII) in the dark (black box). After aeration with 0.001% CO₂ for 6 h, the concentration of aerated CO₂ was switched to 5% for 2 h (V). Red boxes indicate the time of observation. For all culture conditions, the pH of the medium is 7.0. B, Representative LCIB-Clover fluorescence images of C16-B and C16-BC cells. Roman numerals correspond to the culture conditions indicated in A. Ci concentrations in the culture medium were measured using gas chromatography, and 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 C16-BC cells are represented with error bars depicting the interquartile range (n = 11–16). Dunn’s multiple comparisons test was used to assess the statistical significance of LCIB-Clover localization between the different conditions. ^*P-value < 0.05; ^**P-value < 0.01; ^***P-value < 0.001; ^****P-value  < 0.0001, Kruskal–Wallis test with Dunn’s multiple comparison.