Fig 1: MEF2D regulates DYRK1A gene transcription in T98G cells. (A) Genomic organization of DYRK1A isoform 1, 2, 3 and 5 is shown in the scheme. Initiation codon (ATG) was indicated with arrow. E stands for exon. P represents locations of primers for DYRK1A isoforms’ amplification. SC represents stop codons for each isoform. (B) MEF2D expression vector and negative control were transfected into T98G cells. mRNA levels of DYRK1A isoforms were determined by RT-PCR. ß-actin was amplified as internal control. (C) Quantification of B. Values represent means ± SEM; n = 3; *P < 0.01 by Student’s t test. (D) Quantitative fluorescence real time PCR was performed to detect mRNA levels of DYRK1A isoforms in T98G cells transfected with MEF2D and control vector. Values represent means ± SEM; n = 3; *P < 0.01 by Student’s t test. (E) Western blot of DYRK1A isoform 1 and 5 and MEF2D in HEK293 and T98G cells. MEF2D was detected with anti-MEF2D antibody (610774, BD, San Jose, CA, USA). DYRK1A isoform 1 and 5 were detected with anti-DYRK1A antibody (ab156818, Abcam, Shanghai, China). ß-ACTIN was used as loading control.
Fig 2: Confirmation of MRE on DYRK1A promoter by EMSA and ChIP. (A) The consensus MEF2 oligonucleotides were labelled with infrared fluorescence IRDye® 700 and used as probes. EMSA was performed as described in methods. DY-MRE is the oligonucleotide of −282 bp to −251 bp from DYRK1A promoter. Numbers of competitor indicates the molar excess of labelled oligonucleotides. (B) DY-MRE was labelled with infrared fluorescence IRDye® 700 IRDye. EMSA was performed as described in methods. Anti-MEF2D antibody was used in supershift. (C) ChIP was used to confirm the binding of MEF2D with DYRK1A promoter region. Anti-MEF2D antibody, anti-RNA Polymerase II and normal mouse IgG were used in chromatin immunoprecipitation from HEK293 cells. Primers to amplify a short DNA sequence spanning the putative DY-MRE site in DYRK1A promoter region and GAPDH were used for PCR. IgG and H2O were used as the negative controls. (D) Western blot showed that the anti-MEF2D antibody actually immunoprecipitated MEF2D protein. The lower bands were antibody heavy chain.
Fig 3: DYRK1A mRNA expression was correlated with MEF2D in mice neurodevelopment. Quantitative real time PCR was performed to detect total DYRK1A and MEF2D mRNA expression. RNA was isolated from normal mouse brain aging at embryonic days 13.5 (E13.5), embryonic days 18.5 (E18.5), postnatal P1, P7, and P14 and adult. One to three mice were used in each time point as indicated by the numbers after the hyphens. p = 0.0478, r = 0.6182 by Spearman correlation test.
Fig 4: MEF2D specifically activates DYRK1A isoform 5 promoter. (A) The DYRK1A promoter construct pDYluc-long, containing the 1825 bp fragment of 5'-UTR from the human DYRK1A isoform 5, was transfected into HEK293 cells. Dual luciferase activity was measured 48h after transfection by a luminometer. The values represent the means ± S.E. (n = 3); *p < 0.01 by Student’s t test. (B) MEF2D increases DYRK1A promoter activity. The DYRK1A promoter construct pDYluc-long and pGL-3 basic were co-transfected with MEF2D expression plasmid or empty vector into HEK293 cells. Dual luciferase assay was performed 48 h after transfection. Values represent means ± SEM; n = 3; *P < 0.01 by Student’s t test. (C) Knock down of MEF2D leads to decrease of DYRK1A isoform 5 promoter activity compared with control. Dual luciferase assay was performed 48h after transfection. Values represent means ± SEM; n = 3; *P < 0.01 by Student’s t test. (D–F) Overexpression of MEF2D increased DYRK1A isoform 5 protein level in T98G cells. pCMV6-entry-MEF2D and empty control vector were transfected into T98G cells. Cells were harvested after 48 hours’ transfection. MEF2D was detected with anti-MEF2D antibody (610774, BD, San Jose, CA, USA). DYRK1A isoform 5 was detected with anti-DYRK1A antibody (ab156818, Abcam, Shanghai, China). ß-ACTIN was used as loading control. The values represent the means ± SEM; n = 3; *P < 0.01 by Student’s t test. (G–I) The knockdown of si-MEF2D decreased DYRK1A isoform 5 protein level in T98G cells. T98G cells were co-transfected with si-MEF2D and control. Cells were harvested after 48 hours’ transfection. MEF2D was detected with anti-MEF2D antibody. DYRK1A isoform 5 was detected with anti-DYRK1A antibody (ab156818, Abcam, Shanghai, China). ß-ACTIN was used as loading control. The values represent the means ± SEM; n = 3; *P < 0.01 by Student’s t test.
Fig 5: MEF2D regulates DYRK1A kinase activity exemplified by NFATc2 protein expression. (A) MEF2D increased DYRK1A protein and decreased NFATc2. MEF2D expression plasmid and empty vector were transfected into T98G cells with NFATc2 expression vector. NFATc2 was detected with anti-HA antibody. MEF2D was detected with anti-MEF2D antibody (610774, BD, San Jose, CA, USA). DYRK1A isoform 5 was detected with anti-DYRK1A antibody (ab156818, Abcam, Shanghai, China). ß-actin detected by ß-actin monoclonal antibody (SAB1403520; Sigma-Aldrich, Saint Louis, USA) was used as loading control. (B) Quantification of A. Values represent means ± SEM; n = 3; *P < 0.01 by Student’s t test. (C) DYRK1A decreased NFATc2 protein level. DYRK1A expression plasmid and empty vector were transfected into T98G cells with NFATc2 expression vector. NFATc2 was detected with anti-NFATc2 monoclonal antibody (MA1–025, ThermoFisher, Waltham, USA). DYRK1A was detected with anti-DYRK1A antibody (ab156818, Abcam, Shanghai, China). ß-actin detected by ß-actin monoclonal antibody (SAB1403520; Sigma-Aldrich, Saint Louis, USA) was used as loading control. (D) Quantification of C. Values represent means ± SEM; n = 3; *P < 0.01 by Student’s t test.
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