Limb-Bud and Heart (LBH) Upregulation in Cardiomyocytes under Hypoxia Promotes the Activation of Cardiac Fibroblasts via Exosome Secretion

Figure 6

LBH affects TGF-β1 expression and signaling through a positive feedback loop. (a) Protein levels of LBH, CRYAB, TGF-β1, IL-1β, and IL-6 in LBH^KO mouse CFs rescued by lentiviral-induced LBH upregulation (, , and vs. LBH^WT-Lv5NC; and vs. LBH^KO- Lv5NC). (b) In cell western simultaneously testing, the protein levels of TGF-β1 (red) and α-tubulin (green) in LBH^KO mouse CFs transfected with Lv5NC or LBH lentivirus. (c) Secreted TGF-β1 in the cell supernatants of mouse CFs treated with LBH+ exosomes, together with LBH^KO CFs rescued by LBH-overexpressing lentivirus, were measured by ELISAs ( vs. Control/LBH^WT-LvNC; vs. CM-21%-Exo-treated/LBH^KO-Lv5NC). Protein expression of LBH, CRYAB, TGF-β1 (d), and TGF-β1 secretion (e) in mouse CFs after LBH overexpression and subsequent CRYAB knockdown ( and vs. Lv5-NC-Scramble; for Lv5-NC-siCRYAB vs. LBH-siCRYAB; and for LBH-Scramble vs. LBH-siCRYAB). (f) Protein levels of LBH, p-Smad3 and Smad3 in TGF-β1-stimulated mouse CFs with or without treatment with the phosphorylation inhibitor SIS3 ( and vs. Ctrl-0 μM SIS3; , , and vs. TGF-0 μM SIS3).