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. 2022 Feb 8;298(3):101692. doi: 10.1016/j.jbc.2022.101692

Figure 4.

Figure 4

The role of DIMT1 role in 18S methylation and cellular protein expression. Quantification of 18S ribosomal rRNA methylation from total RNA was made by using the qRT-PCR gene expression method (A). The control EndoC-βH1 cells and cells treated with DIMT1 siRNA are shown in the first 4 bars and the last bar represents the positive control, respectively. Data are expressed as mean ± SD (n = 4). Ribosomal RNA expression for 28S, 18S and 5.8S (B); expression of 28S, 18S and 5.8S rRNA in scramble (Sc)- and DIMT1 siRNA-treated rat islets are shown (B). The average rRNA value was normalized by 7SK. Data are expressed as mean ± SD, (n = 4 in rat islets), and were statistically compared with paired Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. RNA Electrophoresis of 28S and 18S rRNA (C); for analysis of 28S and 18S rRNA, 2 μg of total RNA was resolved on denaturing agarose gels and the relative rRNA amount was normalized to total tRNA; the data are expressed as mean ± SD, (n = 4 in rat islets), and were statistically compared with paired Student’s t test; ∗p < 0.05. Protein synthesis detection using OPP (D); the first bar shows the Alexa 568 fluorescence intensity in control and the second and third bar shows the reduced OPP fluorescence in DIMT1 siRNA- and cycloheximide (CHX)-treated cells, respectively. Data are expressed as mean ± SD (n = 3). Physical interaction of NOB1 and PES-1 (E) and their expression in whole-cell lysates with DIMT1 siRNA (F). Western blot and immunoprecipitations are expressed as mean ± SD (n = 3). Proximity ligation assay (PLA; G) for NOB-1/PES-1 interaction is measured by red dot signals that represent the proximity of the NOB-1 and PES-1 proteins. DNA is stained with DAPI. Scale bar: 10 μM. Quantification of the PLA signals per cell after DIMT1 siRNA treatment is shown in the inserted graph (data are mean ± SD; n = 3); statistical comparisons between DIMT1-silenced cells and scramble control cells were made by Student’s t test; ∗∗p < 0.01, ∗∗∗p < 0.001. DIMT1, dimethyladenosine transferase 1; rRNA, ribosomal RNA