Journal: Scientific Reports

Article Title: Angiotensin - converting enzyme Ance is cooperatively regulated by Mad and Pannier in Drosophila imaginal discs

doi: 10.1038/s41598-017-13487-w

Figure Lengend Snippet: SMAD2 and GATA4 are required for ACE expression in HEK293 cells. ( a – c ) Effects of silencing SMAD2 or GATA4 on ACE expression. Y-axis indicates relative expression compared with control siRNA ( siSCR ). ( a ) SMAD2 knockdown results in reduction of ACE level (49.4%). ( b ) GATA4 knockdown reduces ACE level (64.9%). ( c ) Single knockdown of SMAD2 or GATA4 decreases ACE expression (54.1% or 68.9%), respectively. Double siRNAs for SMAD2 and GATA4 result in further inhibition of ACE level (26.5%). ( d ) ACE expression upon overexpressing SMAD2 and/or GATA4 . Double transfection of SMAD2 and GATA4 -expressing vectors induces stronger enhancement in ACE expression compared to single transfection of SMAD2 or GATA4 . ( e ) Enrichment of SMAD2 and GATA4 on ACE promoter. Left: Schematic diagram of human ACE promoter. Transcription start site (TSS) and primer sets used for qPCR are indicated. Right: Relative enrichment of SMAD2 and GATA4 to IgG control. ( f ) Reciprocal co-immunoprecipitation between SMAD2 and GATA4 in HEK293 cells. Overexpressed GATA4 (GATA4-V5) is co-immunoprecipitated with endogenous SMAD2 and vice versa . GATA4 is detected by anti-V5 antibody. ( g ) ACE overexpression suppresses the increased level of phospho-ERK (p-ERK) in SMAD2 / GATA4 -knockdown HEK293 cells. siSMAD2 / siGATA4 in HEK293 cells increases p-ERK compared to siSCR control. ACE overexpression decreases p-ERK in siSMAD2 / siGATA4 -treated cells. ( h ) Angiotensin II (Ang II) treatment reduces the level of p-ERK in SMAD2 / GATA4 -knockdown HEK293 cells. siSMAD2 / siGATA4 in HEK293 cells increases p-ERK compared to siSCR control. Ang II treatment decreases p-ERK in a dose-dependent manner. ( i ) Ang II rescues the reduced cell viability in SMAD2 / GATA4 -knockdown HEK293 cells in a dose-dependent manner. All data represent the mean ± s.e.m. (error bars) and P -values were calculated by using Student’s t -test. n.s, not significant, * P < 0.05. ** P < 0.01. ***P < 0.001. ****P < 0.0001.

Article Snippet: For plasmid transfection, pCEP4 SMAD2 (Addgene, #16484) and tetO-GATA4 (Addgene, #46030) were used. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

Techniques: Expressing, Control, Knockdown, Inhibition, Transfection, Immunoprecipitation, Over Expression