Fig 2: Effects of miR-17-3p and CTR9 on keratinocyte proliferation in vitro. A) miR-17-3p expression levels were measured by RTqPCR 24 h after transfection with miR-17-3p mimic/inhibitor/control. B) The proliferation curves of HaCaT cells after transfection with miR-17-3p mimic/control or miR-17-3p inhibitor/control at 0, 12, 24, 36, 48, and 60 h obtained by CCK-8 assays. C) CTR9 (mRNA) expression levels were measured by RT-qPCR after transfection with siRNA-CTR9/pcDNA 3.1-CTR9/control. D) Proliferation curves of HaCaT cells after transfection with pcDNA 3.1-CTR9/pcDNA3.1/control or siRNA-CTR9/scramble/control at 6, 12, 24, and 36 h by CCK-8 assays; optical density (OD) values at 450 nm of each point were calculated from three independent experiments. Data are presented as mean±SD. *p<0.05; **p<0.01; ***p<0.001.

Fig 3: CTR9 is a direct target of miR-17-3p. A) The potential miR-17-3p seed regions at the 3'UTR of CTR9 mRNA predicted by TargetScan. B) The vector containing wild-type and mutated-type CTR9 3'UTR sequences was constructed. C) The relative luciferase activity of HaCaT cells co-transfected with psiCHECK™-CTR9-WT + miR-17-3p mimic/inhibitor /control or psiCHECK™-CTR9- Mut + miR-17-3p mimic/inhibitor /control; luciferase activity was normalized by the ratio of Renilla luciiferase and firefly signals; the significance of luciferase activity was analyzed using Student' t-test. D) CTR9 mRNA expression was detected following transfection with miR-17-3p mimic/inhibitor/control in HaCaT cells. E) CTR9 protein expression was detected following transfection with miR- 17-3p mimic/inhibitor/control in HaCaT cells. Data are based on three independently performed experiments and are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001.

Fig 4: Upregulation of miR-17-3p in psoriatic skin and its target prediction. A) miR-17-3p expression was determined in the psoriatic skin (n=7) and in the skin of healthy controls (n=6) by RT-qPCR. B) CTR9 mRNA expression was determined in the psoriatic skin (n=8) and healthy control skin (n=8) by RT-qPCR. (C) CTR9 protein expression levels are shown with the mean optical density (MOD) in psoriatic skin (n=7) and healthy control skin (n=10) by immunohistochemistry; the expression levels of miR-17-3p and CTR9 were calculated using the 2-ΔΔCt method. Data are based on three independently performed experiments and are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001.

Fig 5: RNMT-RAM Interacts with Transcription-Associated Complexes(A) Identification of HA-RNMT-interacting proteins using SILAC-based mass spectrometry.(B) Fold enrichment of proteins in HA-RNMT IP/IgG IP plotted against the −log10 posterior error probability score. Transcription-associated proteins are highlighted (with the number of peptides identified in parentheses). Three-fold enrichment over control cutoff.(C–E) HeLa cell extracts subject to IP-western blot analysis for CTR9 (C), RNMT (D), and LEO1 (E). Where indicated, IPs were incubated with or without RNase (C). Anti-glutathione S-transferase (GST) IP, negative control.(F) HeLa cells transfected with pcDNA5 HA-RNMT WT and mutants or vector control for 48 hr. Extracts were subject to CTR9 IP and HA western blot analysis.(G) HeLa cells transfected with RAM siRNA or non-targeting control for 36 hr. CTR9 ChIP was performed. Average and SEM are presented for the PCR signal relative to input (background subtracted) (n ≥ 3).(H) HeLa cells transfected with two RAM siRNAs or sc siRNA for 24 and 36 hr. Western blot analysis is shown.