Fig. 1: METTL16 suppresses KSHV lytic replication. | Cell Death & Disease

Fig. 1: METTL16 suppresses KSHV lytic replication.

From: METTL16 controls Kaposi’s sarcoma-associated herpesvirus replication by regulating S-adenosylmethionine cycle

Fig. 1: METTL16 suppresses KSHV lytic replication.

A iSLK-RGB-BAC16 cells were transfected with siRNAs targeting METTL16 (siM16-1 and siM16-2) or the control siRNA (siCtrl) for 48 h. RNA or protein were harvested, and analyzed by RT-qPCR or Western-blotting to monitor the knockdown efficiency. GAPDH was used as normalization control. B, C iSLK-RGB-BAC16 cells were transfected with siRNAs targeting METTL16 or the control siRNA for 24 h. Cells were treated with 3 mM of sodium butyrate (NaB) for 48 h and observed with a fluorescent microscope (B) and analyzed by flow cytometry for EGFP-positive cells (C). D iSLK-RGB-BAC16 cells transfected with siRNAs targeting METTL16 or the control siRNA were treated with 3 mM of NaB for 72 h and examined for the expression level of KSHV transcripts RTA, ORF57, ORF-K8 and ORF65 by RT-qPCR. GAPDH was used as normalization control. E iSLK-RGB-BAC16 cells transfected with siRNAs targeting METTL16 or the control siRNA were treated with 3 mM of NaB for 72 h. Cells were lysed and analyzed for the expression of RTA, ORF57, K8, and ORF65 proteins by Western-blotting. GAPDH was used as a loading control. F, G iSLK-RGB-BAC16 cells transfected with siRNAs targeting METTL16 or the control siRNA were treated with 3 mM of NaB for 48 h. The cells were then washed with PBS and cultured in fresh medium without NaB, hygromycin, puromycin and G418 for 72 h. Supernatants were collected and used to infect MM cells. mRFP1-positive cells were imaged (F) and quantified at 48 h post-infection (G). H iSLK-RGB-BAC16 cells were transfected with a plasmid expressing METTL16 or a vector control. RNA and protein were harvested and analyzed by RT-qPCR and Western-blotting to monitor the overexpression efficiency. GAPDH was used as a normalization control. I iSLK-RGB-BAC16 cells with overexpression of METTL16 were treated with or without 3 mM of NaB for 72 h. Levels of KSHV transcripts RTA, ORF57, PAN RNA and ORF65 were analyzed by RT-qPCR. *, **, and *** indicate p values of <0.05, <0.01, and <0.001, respectively, ns not significant.

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