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. 2023 Oct 11;10(32):2302231. doi: 10.1002/advs.202302231

Figure 6.

Figure 6

Downregulated ALDH2 upregulates ELK3 expression through LIN28B‐mediated mRNA stability. A) Luciferase assay exhibiting relationship between ALDH2 and ELK3 promoter (n = 4 per group). B) The ELK3 mRNA decay in HUVECs was evaluated using RT‐qPCR following pretreatment with actinomycin D to inhibit transcription (n = 6 per group). C) Potential proteins mediating the ELK3 mRNA stability by the RNA‐protein interaction database. D) RT‐qPCR of ELK3 in HUVECs treated with LIN28B siRNA (n = 4 per group). E) Representative immunoblots and quantification for LIN28B and ELK3 in LIN28B siRNA‐treated HUVECs (n = 3 per group). F) RT‐qPCR was utilized to determine ELK3 mRNA decay in HUVECs after pretreatment with actinomycin D to inhibit transcription (n = 4 per group). G) RT‐qPCR of ALDH2 and LIN28B in HUVECs treated with a flag‐ALDH2 plasmid (n = 4 per group). H) Representative immunoblots and quantification for ALDH2 and LIN28B in flag‐ALDH2 plasmid‐transfected HUVECs (n = 3 per group). I) Visualization of ALDH2‐ LIN28B interacting interface prediction based on the Hdock tool. Cartoon representation depicts the complex structures with the predicted interaction residues. J) Co‐immunoprecipitation assay of ALDH2 and LIN28B in HUVECs (n = 3 per group). K) Flag‐ALDH2 was co‐transfected with HA‐LIN28B into HEK293T. Cell lysates were subjected to Co‐IP with an anti‐Flag antibody, followed by western blotting (n = 3 per group). L) Representative confocal microscopic images of colocalization of ALDH2 and LIN28B in Hela cells. Scale bar, 25 µm. M) Co‐immunoprecipitation assay of ALDH2 and LIN28B in HUVECs cell line treated with Ang II (n = 3 per group). N) RNA immunoprecipitation assay was performed using HUVECs cell lysates. RT‐qPCR was performed to measure relative LIN28B binding to ELK3 mRNA using primers designed for ELK3. Samples immunoprecipitated with immunoglobulin G (IgG) served as control (n = 4 per group). O,P) Representative immunoblots and quantification for vinculin, JAM‐A, VE‐cadherin, p120‐catenin, claudin‐5, LIN28B, and ALDH2 in HUVECs treated with shALDH2 or LIN28B siRNA after 24 h Ang II treatment (n = 4 per group). Q) VE‐cadherin‐specific antibody staining (green) in HUVECs treated with shALDH2 or LIN28B siRNA after 24 h Ang II treatment (n = 3 per group). Scale bar, 25 µm. R) Schematic diagram of the regulation mechanism of ELK3 mRNA stability. Data are presented as mean ± SEM. Unpaired two‐tailed Student's t tests with Welch's correction were used in (A,G) and unpaired two‐tailed Student's t test was used in (H). Two‐way ANOVA followed by Bonferroni post hoc analyses were applied in (B,F,N,P). One‐way ANOVA followed by Bonferroni post hoc tests were applied in (D,E). A.U. indicates arbitrary unit.