M6PR-CI is required for PS-ASO activity in mouse cells and in mice. (A) qRT-PCR quantification of M6PR-CD and M6PR-CI mRNA levels in mouse MHT cells at 72 h after transfection with control siRNA or siRNAs targeting M6PR-CD or M6PR-CI mRNA. Error bars are standard deviations from three independent experiments. (B) Western analysis of M6PR-CI protein in MHT cells transfected with indicated siRNAs. (C) Immunofluorescent staining of M6PR-CD in MHT cells treated with control or M6PR-CD-targeted siRNAs. Scale bars, 10 μm. (D) qRT-PCR quantification of SRB1 mRNA in MHT cells pretreated with siRNAs, followed by incubation with SRB1-targeting PS-ASO 353382. Error bars are standard deviations from three independent experiments. P-values were calculated based on F-test. (E) Upper panel: Quantification of M6PR-CD and M6PR-CI mRNAs in livers of mice treated with control PS-ASO 716837, PS-ASO 1332272 targeting M6PR-CD, or PS-ASO 1332273 targeting M6PR-CI. These mice were subsequently treated with the SRB1 ASO at different doses, as in panel F. Error bars are standard deviations from three animals. Lower panel: M6PR-CI protein levels evaluated by western in livers of mice treated with indicated control or M6PR PS-ASOs. The numbers above lanes represent different animals in each group. (F) qRT-PCR quantification of SRB1 mRNA in livers of mice treated with control PS-ASO 716837, M6PR-CD-targeted PS-ASO 1332272, or M6PR-CI-targeted PS-ASO 1332273 for 5 days followed by treatment with SRB1 PS-ASO 353382 at indicated doses for 24 h. Error bars are standard deviations from three animals. P-values were calculated based on unpaired t-test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (G) qRT-PCR quantification of PTEN mRNA in livers of mice treated with control PS-ASO 716837, PS-ASO 1332272 targeting M6PR-CD or PS-ASO 1332273 targeting M6PR-CI for 5 days, followed by treatment with PTEN PS-ASO 116847 at indicated doses for 24 h. Error bars are standard deviations from three animals. P-values were calculated based on unpaired t-test. *, P < 0.05; ***, P < 0.001.