Fig. 5

LNMAT1 forms a triplex with the CCL2 promoter and interacts with hnRNPL. a FISH analysis of the subcellular distribution of LNMAT1 in UM-UC-3 cells. Scale bar: 10 μm. b Nuclear fractionation analyses and qRT-PCR analyses of LNMAT1 expression in the nucleus and cytoplasm. c Transcriptional activity of the CCL2 promoter was evaluated using sequential deletions and by examining the CCL2 promoter linked to Renilla luciferase activity. Two-tailed t-tests was used. d Schematic presentation of the potential LNMAT1 binding sites in the CCL2 promoter. e, f ChIRP analysis of LNMAT1-associated chromatin in UM-UC-3 cells. Retrieved chromatin was quantified by qRT-PCR. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s tests for multiple comparison. g–i Fluorescence resonance energy transfer (FRET) of a 5:1 mixture of TFO in LNMAT1 with TTS in the CCL2 promoter sequences is shown in red. The individual TFO is blue and TTS is shown in black (g, h). FENDRR/PITX2 was used as the positive control (i). j–l Circular dichroism (CD) spectroscopy of a 1:1 mixture of TFO in LNMAT1 with TTS in the CCL2 promoter sequences is shown in blue. The sum of individual TFO and TTS is shown in red (j, k). FENDRR/PITX2 was used as the positive control (l). m An RNA pull-down assay was performed using LNMAT1 sense and antisense RNAs in UM-UC-3 cells, followed by silver staining. A red arrow indicates hnRNPL. n, o The interaction between LNMAT1 and hnRNPL was confirmed by RNA pull-down and western blotting with nuclear extract or purified recombinant hnRNPL. p RNA immunoprecipitation (RIP) analysis using the anti-hnRNPL antibody revealed that LNMAT1 interacted with endogenous hnRNPL in UM-UC-3. U1 was used as the negative control. Statistical significance was assessed using Two-tailed t-tests was used. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01