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. 2023 Feb 22;14:977. doi: 10.1038/s41467-023-36571-4

Fig. 3. The micropore mediates salvage of host cytosolic materials and Golgi ceramide.

Fig. 3

a–e The key micropore proteins are involved in GFP acquisition from host cell. a The parasite lines were grown in GFP-expressing host cells for observation and counting of GFP+ and GFP− parasites. Percentages of GFP+ parasites in population were plotted for three independent experiments with triplicates (n > 100 parasites for each replicate). b, c Band intensities of Western blots with AID-K13 induced for 2, 4 and 6 h were normalized to those at 0 h (N = 4). d, e In GFP acquisition assays, LHVS (10 μM) was added, while the IAA induction started for the induced groups, as illustrated (d), three independent experiments were performed (n > 200 for each replicate). Scale = 2 µm. f, g Western blot detection of biotin levels in parasites, showing streptavidin Li-COR IRDye 800CW (Strep-800CW) bands on blots (green) (f). The intensities of ACC1 (288 kDa) and PC (152 kDa) were normalized to those at 0 h (−IAA) (g). h, i IFA detection of biotin levels, showing streptavidin Alexa Flour-488 (Strep-488) (green) signal in the AID lines grown in IAA, and lines grown in the vehicle (−IAA) (h). ACP was the apicoplast marker. The intensities of vacuoles (8 and 16 parasites) were measured (n = 48), and normalized to the control (−IAA), and plotted as ratio (i). Scale = 5 µm. j, k Detection of ceramide salvage by pulsing parasites with the probe, showing representative images of the pulsing with BODIPY TR Ceramide for 30 min in parasites grown in auxin for 36 h (j).The staining foci numbers and fluorescence in vacuoles were measured automatically by the software system (N ≥ 50 for each independent experiment) (k). Scale = 10 µm. Three independent experiments and triplicates were performed, and a, c, e, g, i, k Mean ± SD, and analyzed by two-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001 and other exact p values with significance are provided. b, c, f, g The samples were derived from the same experiment and blots were processed in parallel with the loading control on the same blots.