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. 2022 Sep 1;11(17):2724. doi: 10.3390/cells11172724

Figure 3.

Figure 3

Statistical analysis revealed alteration of peroxisomal content by light conditions. (A) Schematic of fluorescently labeled palmitic acid (FL-C16) pulse experiment. C. reinhardtii expressing both CFP-SLI and CIS2-RFP was cultured in TAP with FL-C16 for 8 h, then transferred to a fresh medium under four different conditions for 24 h before imaging: TAP with continuous light, minimal medium (without acetate, MM) with continuous light, TAP in the dark, and MM in the dark. (B) Localization of FL-C16 and peroxisomal marker proteins after 24 h in each condition. Thirty optical sections of each 20 µm z-stack are projected into one image with their maximum intensity. Co-localizations of all three markers are rarely observed, indicated by white arrows. (C,D) Thirty optical sections within the 20 µm z-axis were obtained by confocal microscopy. Colocalization in the optical sections was analyzed with ComDet (ImageJ plugin for analyzing colocalization of bright intensity spots). Mean of the spot number, standard error of the mean, numbers of cells, and optical sections for the analysis are, TAP with continuous light (TAP Light: mean 9.7, S.E. 0.47, 8 cells, 20 sections), MM with continuous light (MM Light: mean 5.75, S.E. 0.46, 7 cells, 32 sections), TAP in the dark (TAP Dark: mean 12.1, S.E. 2.78, 3 cells, 6 sections), and MM in the dark (MM Dark: mean 6.94, S.E. 0.76, 5 cells, 19 sections). (C) Compartments detected in an optical section are reduced when the cells are cultured without acetic acid. A total number of compartments in an optical section detected by ComDet is presented as a box plot with individual data points. Notice that the number of compartments is reduced when the cells are cultured without acetic acid (MM) regardless of light conditions. A letter on the top of each bar indicates mean comparisons for each pair using Student’s t-test. Levels not connected by the same letters are significantly different (p < 0.05). (D) Compartments change contents depending on culture conditions. Colocalization rates in the compartments are calculated, based on the culture conditions (TAP Light: n = 194, TAP Dark: n = 105, MM Light: n = 73, MM Dark, n = 132).