Fig. 5: SERCA2a is targeted to and degraded in lysosomes via autophagy in TMAO-induced cardiac hypertrophy.

a Representative images of immunofluorescence detection of LC3 (red) and SERCA2 (green) in H9c2 cells treated with TMAO (1 mM) for 24 h, followed by treatment with BafA1(10 nM) for 24 h, n = 3. Scale bar is 10 μm. b–f Western blot analysis of LC3B2, ATG5, ATG7and p62 after 48 h of TMAO treatment, n = 3. g-i Western blot analysis of LC3B2 and p62 in H9c2 cells pretreated with 3-MA (5 mM) for 1 h, then exposed to TMAO(1 mM) for another 48 h, n = 3. j, k Representative images of autophagosomes detected by transmission electron microscopy in H9c2 cells pretreated with 3-MA (5 mM) for 1 h, then exposed to TMAO(1 mM) for another 48 h. red arrow: autolysosome; yellow arrow: autophagosome. l–n Representative images of fluorescence detection of H9c2 cells transfected with a mCherry-EGFP-LC3 reporter, followed by treatment with 1 mM TMAO for 48 h, scale bar is 20 μm, *P < 0.05, **P < 0.01. o–u Western blot analysis (n = 3) of SERCA2a, ANP, and MYH7 and representative microscopic images of cells with rhodamine-phalloidin staining of the cytoskeleton and DAPI staining of the nucleus and cell surface area analysis (n = 4) in H9c2 cells were pretreated with 3MA (5 mM) for 1 h, followed by treatment with TMAO (1 mM) for another 48 h, scale bar is 40 μm. Statistical analyse was performed using the One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent S.E.M.