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. Author manuscript; available in PMC: 2024 Jul 11.
Published in final edited form as: Cell Metab. 2023 Jun 15;35(7):1163–1178.e10. doi: 10.1016/j.cmet.2023.05.010

Figure 4. ACSS2-generated Ac-CoA regulates TGFβ signaling.

Figure 4.

A-B, Cytosolic and nucleic R-SMADs examined by western blotting (A) and immunostaining (B) in HUAECs treated with Scramble siRNA, Scramble siRNA + TGFβ2, or ACSS2 siRNA + TGFβ2 respectively. Scale Bar: 25 μm. C, Representative blots of nucleic R-SMADs in HUAECs treated with Scramble siRNA, ACLY siRNA, or ACSS2 siRNA separately. D, Representative blots of acetylated R-SMADs in HUAECs transduced with Scramble siRNA or ACSS2 siRNA. Acetylated proteins were captured using agarose beads conjugated with anti-Acetylated lysine antibody, and acetylated R-SMADs (Ac-SMAD2/3/4) were detected using anti-SMAD2/3 antibody and anti-SMAD4 antibody. E, Blots showing the expression of ALK5 in HUAECs transduced with Scramble siRNA or ACSS2 siRNA in the presence of TGFβ2 for 7 days. F, mRNA level of ALK5 in HUAECs under different conditions: Scramble siRNA, Scramble siRNA + TGFβ2, or ACSS2 siRNA + TGFβ2 respectively. G, Blots of ALK5 level in HUAECs following with SMAD2 deficiency for 4 days. H, Blot showing ALK5 level in HUAECs transduced with Scramble siRNA or SMAD4 siRNA for 4 days. I, Representative blots of V5-tagged ALK5 (ALK5-V5) protein level following gradient overexpression of ACSS2-HA in HUAECs using adenoviral strategy(i). J-K, Representative blots of ALK5-V5 in HUAECs transfected with Scramble siRNA or ACSS2 siRNA(J) / PDK4 siRNA(K) separately for 4 days. ALK5-V5 was adenoviral overexpressed in HUAECs on the next day after siRNA transfection.