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Cell Host Microbe. Author manuscript; available in PMC 2017 Dec 14.
Published in final edited form as:
Cell Host Microbe. 2016 Dec 14; 20(6): 785–797.
doi: 10.1016/j.chom.2016.11.001
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Figure 3

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CRISPRi against MLST8 in latent K562 cells prevents reversal of HIV latency by LRAs

(A) Procedure to obtain latent CRISPRi K562 cells and transduce them with sgRNA lentiviruses and select by puromycin. LRAs were added to test reactivation of HIV.

(B) Efficiency of MLST8 knockdown with three different sgRNAs checked by western blot. Cells transduced with NC (negative control) sgRNA lentiviruses done in duplicate (NC-1 and NC-2) were used as control.

(C, D) Percentage of GFP-positive cells 24 h after reactivation with PMA (C) and Ingenol-B (D). Data are represented as mean +/− SD of triplicate values, representative of two independent experiments.

(E) Simple scheme representing the mTORC1 and mTORC2 subunits and regulator that were knockdown by CRISPR interference.

(F,G,H,I) Latent CRISPRi K562 cells were transduced with sgRNA lentiviruses targeting MTOR (F), RICTOR (G), RAPTOR (H), TSC1 (I) and selected by puromycin. Percentages of GFP-positive cells 21–24 h after reactivation with PMA are indicated on the upper panels. Efficiency of knockdown for each gene is shown on the lower panels with western blot. Cells transduced with NC (negative control) sgRNA lentivirus were used as control. Data are represented as mean +/− SEM of at least three independent experiments.

See also Figure S1

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