C10329

Ring stage parasites at a parasitemia of about 5% (3D7), (15 hours post-invasion) were incubated at a final concentration of 0.5 mM 5-ethynyl uridine (EU) for 30 h. EVs were isolated from the conditioned medium as described above with exception of the sucrose gradient. The cells and EVs were subsequently fixed with 4% para-formaldehyde and permeabilized with 0.1% Triton-X-100. EU incorporated EV RNA was detected using Click chemistry according to the manufacturer’s protocol (Invitrogen, C10329) and nuclei were counterstained using Hoechst 33342 and analyzed with a MACSQuant VYB flow cytometer. Flow cytometry data acquisition was performed using FlowJo X (Tree Star, Ashland, OR).

Both repair proficient BEAS-2B and NER deficient (XPA knock-down) cells were grown in a 4-well chamber slide and exposed to NNA (10 μM) or mock exposure for 24 h. At the end of exposure both mock and NNA-exposed samples from BEAS-2B and siXPA down-regulated BEAS-2B cells were proceed for EU labeling as described above. For recovery of RNA synthesis assay both BEAS-2B and siXPA down-regulated cells were washed with PBS after 24 h exposure to 10 μM NNA were allowed to repair for another 24 h with complete DMEM (10% FBS) medium followed by EU incorporation and visualization by Click IT conjugation of Alexa Fluor 488 according to the manufacturer’s protocol (Invitrogen, Cat# c10329) and the method in [9 (link)]. Images were obtained and quantified by Image J as described above. For each experiment, >25 randomly selected cells from 40x magnification were used for analysis.

For drug-treated groups, embryos were placed to G-1 PLUS medium supplemented with 0.05% DMSO, 100 μM α-amanitin (Sigma-Aldrich, A2263), 3 μg/mL aphidicolin (Sigma-Aldrich, A4487) or 1 μM JQ1 (MedChem Express, HY-13030), respectively, after ICSI-induced fertilization. For Hdac overexpression, the mRNAs of Hdac1 and Hdac2 were synthesized as described above and mixed at a concentration of 500 ng/μL each, and the Hdac mRNA mixture was injected into MII oocytes before ICSI. Embryos injected with water served as the control group for Hdac overexpression. At 6 hpf or 12 hpf, the parental PN were collected for low-input MNase-seq.
To verify that the transcription or DNA replication activities were successfully inhibited in each group, we transferred embryos into G-1 PLUS medium supplemented with corresponding drugs as well as 1 mM EU or 10 μM EdU at 8 hpf, and cultured these embryos for another 4 h. At 12 hpf, we fixed the embryos with 4% paraformaldehyde in PBS and performed EU (Invitrogen, C10329) or EdU staining (Invitrogen, C10634) following the manufacturer’s instructions, respectively.