Alexa fluor 488 alkyne

Parasites were washed with PBS three times and 1 x 10⁷ cells were incubated at 37°C in the case of ICA and TCT, and at 28°C in the case of Epi, in 1 mL of 2% delipidated BSA-DMEM. After 30 min, 100 μM Click-IT myristic acid, azide (Life Technologies, Thermo Fisher Scientific) was added from 50 mM stock solutions in dimethyl sulfoxide (DMSO). The same volume of DMSO was used as a negative control. Parasites were further incubated for 6 h. For confocal microscopy analysis, they were processed as described above. After permeabilization (0.1% Triton X-100 in PBS for 10 min, at room temperature), cells were washed three times with 3% BSA in PBS. “Click” reaction between the myristic acid azide and Alexa Fluor 488 alkyne (Invitrogen) was performed according to the manufacturer instructions using the Click-iT Cell Reaction Buffer Kit (Invitrogen). Parasite DNA was labeled with DAPI. Samples were visualized and images acquired as described above.

To determine nascent protein synthesis, Click-iT AHA (l-azidohomoalaine), Alexa Fluor 488 alkyne, and Click-iT cell reaction buffer kit were purchased from Invitrogen and used according to the manufacturer’s protocol. Briefly, Click-iT AHA (50 µM), a methionine analog containing an azide moiety, was added to cells in methionine-free medium (Invitrogen) for 1 h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde, and permeabilize with 0.25% Triton X-100. Detection of the incorporated amino acid utilizes a click reaction between an azide and alkyne, where the azido-modified protein is detected with an Alexa Fluor® 488 by flow cytometry.

Bs and Bm cells were grown over night at 30 °C in S7 minimal medium^{38 (link)}, containing all amino acids or lacking Met. Cells were then diluted to OD₆₀₀ 0.05 and incubated in S7 medium at 37 °C to OD₆₀₀ 0.3. Next, cells were washed in PBSx1 to remove residual Met, diluted to OD₆₀₀ 0.05, and incubated in S7 medium lacking Met, either in monoculture or co-culture, and supplemented with AHA (1 mM). Cells were grown until reaching OD₆₀₀ 0.15 (~ 1.5–2 h), and click reaction was carried out. In brief, cells were centrifuged and washed in PBS×1 to remove residual AHA, resuspended in 50% ethanol for 3 min, followed by a wash in 100% ethanol. Cells were then centrifuged and re-suspended in 230 µl PBSx1 and 12.5 µl of freshly made sodium ascorbate (100 mM; Sigma). Then, mixed separately with 2.5 µl of 50 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, Sigma), 1.25 µl of 20 mM CuSO₄ (Sigma), and 3 µl of 2 mM alkyne dye (Alexa Fluor 488 Alkyne, Invitrogen), and left to react for 3 min in the dark at RT. The mixture was added to the cells, and samples were left in the dark at RT for 30 min. Finally, Cells were centrifuged, washed in PBSx1, and observed by fluorescence microscopy.

NHS-Azide-injected samples were processed for click chemistry according to the following protocol, modified from previous studies (Dieterich et al., 2010 (link); Hinz et al., 2012 (link)). Sections or wholemounts were transferred to Eppendorf tubes or six-well plates with reaction mixture composed of 100 μm tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Sigma) dissolved in 4:1 tBuOH/DMSO (Sigma), 100 μm CuSO₄ (Sigma), 1.25 μm Alexa Fluor 488 alkyne (Invitrogen), and 250 μm tris(2-carboxyethyl)phosphine (TCEP, Sigma). The reaction proceeded overnight at room temperature.

Cultured cells were labeled with Cy5-conjugated puromycin (5 uM) for one hour at the end of the indicated time point following drug treatment. Cy5-conjugated puromycin is readily incorporated into live cells without the need for methionine starvation. Changes in mean fluorescence intensity of Cy5-conjugated puromycin as a measure of newly produced protein were analyzed by flow cytometric analysis. For nascent protein labeling in 293T cells, we used Click-iTR AHA (L-azidohomoalanine) metabolic labeling reagent purchased from Invitrogen (cat no. C10102) and following the manufacturer’s instructions. Briefly, cells were incubated in a methionine-free medium for 30 min before AHA labeling for 1 h after doxycycline treatment (2 mg/mL). Cells were fixed with 4% paraformaldehyde (wt/vol) in PBS for 15 min and permeabilized with 0.25% Triton X-100 (vol/vol) in PBS for 15 min followed by one wash with 3% BSA. Cells were then stained using Alexa Fluor 488 Alkyne (Invitrogen cat no. A10267) using Click-iT Cell reaction Buffer Kit (Invitrogen cat no. C10269). Changes in mean fluorescence intensity (MFI) of Alexa Fluor 488 Alkyne staining were detected by flow cytometric analysis and used as a measure of newly synthesized protein.

Glucose was conjugated to Alexa Fluor 647 or Alexa Fluor 488 using CuAAC click chemistry. Solutions were prepared containing 1X PBS, 30 μL of a pre-prepared mixture of 0.1 M CuSO₄/0.2 M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 30 mM azido-PEG4-β-D-glucose (BroadPharm), 50 μL of a 10 mg/ml stock of either Alexa Fluor 647 alkyne or Alexa Fluor 488 alkyne (Invitrogen), and H₂O to a final volume of 225 μL. 25 μL of a freshly-prepared 50 mM solution of sodium ascorbate was added to the reaction mixture for a final concentration of 5 mM. The solution was degassed using N₂ for 5 minutes, then reacted for 1 hr at room temperature with rotation. After the labeling reaction was complete, the samples were dialyzed in water using Tube-O-DIALYZER Micro 1 K MWCO (G-Biosciences).