Eukaryot Cell 8:520C529. Values are the mean the standard error of the mean of three impartial experiments in which least 250 vacuoles were counted. Download Physique?S1, TIF file, 0.04 MB mbo005152521sf1.tif (42K) GUID:?F71BCE65-943B-4B1D-A5A7-7801A1D547C4 Physique?S2&#x000a0: Growth of TgATG8-depleted parasites is not affected before egress from your first host. TATi1-Ku80 and cKd-TgATG8 parasites were produced in their host cells with or without ATc. After 24?h, the number of Betamipron parasites per vacuole was determined. Values are the mean the standard error of the mean of three impartial experiments, where 200 vacuoles were counted for each condition. Download Physique?S2, TIF file, 0.1 MB mbo005152521sf2.tif (62K) GUID:?04E387D7-0301-4BD0-A8FD-EDCC84E6065E Physique?S3&#x000a0: Tomato- and GFP-fused TgATG8 can associate with membranes as native TgATG8. Protein lysates from parasites expressing Tomato-TgATG8, GFP-TgATG8, or mutated versions of these proteins where glycine 124 was replaced with an alanine (G124A, abrogates the ability to be conjugated to lipids) were separated by SDS-PAGE in the presence of 6?M urea and analyzed by Western blotting. The faster-migrating form corresponds to the Betamipron PE-conjugated, and thus membrane-bound, form of the protein, as (i) it is absent from your G124A cell lines and (ii) conjugation is usually induced by starvation in HBSS medium like native TgATG8, although in that case, membrane recruitment seems less efficient for Tomato-TgATG8. Download Physique?S3, TIF file, 0.1 MB mbo005152521sf3.tif (91K) GUID:?321776A7-94B7-4DDE-9DDE-7EE622F9245C Physique?S4&#x000a0: Absence of apicoplast DNA and stromal marker TgCPN60 indicates complete Betamipron organelle loss in TgATG8-depleted parasites. Shown is an immunofluorescence analysis of HFF monolayers infected for 24?h in the presence of ATc by parental TATi1-Ku80 and mutant cKd-TgATG8 parasites, stained for peripheral apicoplast protein TgATRX1, analyzed together with stromal protein TgCPN60 (A) and DAPI (B). Level bars, 5?m. Download Physique?S4, TIF file, 0.2 MB mbo005152521sf4.tif (230K) GUID:?D78D6307-2A2E-4854-BAD9-1D9E829A52E5 Figure?S5&#x000a0: Apicoplasts found in the residual bodies of TgATG8-depleted parasites appear normal. Shown is usually a vacuole made up of TgATG8-depleted tachyzoites produced for 48?h with ATc with an apparently intact apicoplast in the residual body (a magnified view is presented on the right). Scale bars: left image, 2?m; right image, 200?nm. Download Physique?S5, TIF file, 0.6 MB mbo005152521sf5.tif (625K) GUID:?8A352E18-8145-43B7-AEE1-56DC32520827 Physique?S6&#x000a0: Most organelles are unaffected by TgATG8 depletion. Organelles were analyzed by IFA in intracellular tachyzoites of the parental cell collection and the TgATG8 conditional knockdown after 3?days of incubation with ATc. (A) Mitochondria were labeled with anti-TgHSP60 antibody (top), and the proportion of parasites displaying a fragmented mitochondrion was quantified (bottom). (B) Micronemes were labeled with anti-TgAMA1 antibody. (C) The IMC was labeled with anti-TgIMC1 antibody. (D) Dense-granule secretion was assessed with an anti-TgGRA3 antibody, and rhoptries were labeled with an anti-TgRON4 antibody. Level bars, 5?m. Download Physique?S6, PDF file, 0.3 MB mbo005152521sf6.pdf (364K) GUID:?BF5F9389-E640-4219-973B-5CD97E435001 Physique?S7&#x000a0: The Golgi apparatus remains associated with the centrosome during the division of TgATG8-depleted parasites. An immunofluorescence analysis of cKd-TgATG8 intracellular tachyzoites after 24?h days of ATc incubation is usually shown. The apicoplast and centrosomes were labeled with anti-TgATRX1 and anti-centrin1 antibodies, respectively; the Golgi apparatus was labeled by transient Betamipron expression of the RFP-fused GRASP protein. Scale bar, 5?m. Download Physique?S7, TIF file, 0.1 MB mbo005152521sf7.tif (84K) GUID:?93287327-271A-44AF-9E97-ECC24D6EBEDC Movie?S1&#x000a0: GFP-TgATG8 is recruited to the apicoplast prior to the formation of child cell scaffolds. Time-lapse imaging of the IMC (labeled with TgIMC1-Tomato) and GFP-TgATG8 during two successive rounds of division is shown. Download Movie?S1, AVI file, 3.6 MB mbo005152521sm1.avi (3.7M) GUID:?429E4DE2-FD1F-453F-8A1C-8E602D6A49E3 Movie?S2&#x000a0: GFP-TgATG8 is recruited to the apicoplast after division of Betamipron the Golgi apparatus. Time-lapse imaging of the Golgi apparatus SPARC (labeled with GRASP-RFP) and GFP-TgATG8 is usually shown. Download Movie?S2, AVI file, 0.7 MB mbo005152521sm2.avi (728K) GUID:?CA0BE74C-31C5-4416-BA2C-F1ACA240D2A5 ABSTRACT Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through.