Ab280888

Astragaloside IV (C₄₁H₆₈O₁₄, A800922, purity ≥98.5%) was purchased from Maclin Inc. and the chemical structure is shown as Figure 1A. ROS fluorescent probe‐DHE (R001, Vigorous Biotechnology) used for detection of ROS in tissues and DCFH‐DA (KM0062) used for detection of cellular ROS was purchased from Beijing Biolab Technology Co. The antibodies used in this study including NRF1 (ab175932, abcam), PGC‐1α (ab106814, abcam), TFAM (ab252432, abcam), Caspase3 (9662, CST), cleaved‐caspase3 (ab32042, abcam), Bax (ab111391, abcam), Bcl2 (2872, CST), α‐SMA (A5228, sigma), p‐smad2 (ab280888, abcam), smad2 (ab40855, abcam), MT‐CO1 (ab203912, abcam), MT‐ND6 (PA5‐109993, Invitrogen) and MT‐ATP6 (PA5‐116789, Invitrogen). MG‐132 (M7749) and Cycloheximide (C7698) were purchased from Sigma.

Fresh heart tissue was added to RIPA lysis buffer to obtain total protein. Protein (25 μg) was subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with skim milk for 30 min at room temperature and then incubated with primary antibodies at 4°C overnight. The next day, after washing with TBST, the membranes were incubated with conjugated secondary antibodies (1:3000, CST) at room temperature for 1 h, and the bands were visualized by chemiluminescence. The main antibodies used in this study were: anti-TGF-β (ab215715, Abcam), anti-p-Smad2 (ab280888, Abcam), anti-Smad2 (ab33875, Abcam), anti-p-AKT (ab38449, Abcam), anti-AKT (orb11276, Biorbyt), anti-p-ERK1/2 (ab214036, Abcam), anti-ERK1/2 (ab184699, Abcam), anti-p-IKKα (ab138426, Abcam), anti-IKKα (ab32041, Abcam), anti-p-P65 (ab76302, Abcam), anti-P65 (ab16502, Abcam), and anti-GAPDH (ab8245, Abcam).

Chemiluminescent based on horseradish peroxidase (HRP) were used to detect and visualize western blots. The following antibodies were used: anti-CPT1α (12252S, Cell Signaling Technology, Beverly, MA, USA), PACS2 (GTX17244, GeneTex, Irvine, CA, USA), VE-cadherin (ab33168, Abcam), β‐actin (A5441, Sigma‐Aldrich), AKT (2938S, Cell Signaling Technology), p-AKT (9018S, Cell Signaling Technology), Smad2 (5339T, Cell Signaling Technology), p-Smad2 (ab280888, Abcam, UK), PPARα (ab215270, Abcam, UK), and HRP conjugated goat anti-rabbit IgG secondary antibody (AS09 602, Agrisera, Vannas, Sweden).

Liver tissues and HSC-T6 cells were lysed with RIPA buffer (#ab156034, Abcam, Cambridge, UK) containing a phosphatase inhibitor and PMSF (#ab141032, Abcam, Cambridge, UK). Protein samples were denatured and separated by 10–15% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked and incubated with primary antibodies. The primary antibodies were as follows: collagen I (1:1000; #ab270993, Abcam, UK), TGF-β1 (1:500; #sc-130348, Santa Cruz, CA, USA), collagen III (1:1000; #ab184993, Abcam, UK), Smad2 (1:500; #sc-393312, Santa Cruz, CA, USA), Smad3 (1:500; #sc-101154, Santa Cruz, CA, USA), p-Smad2 (1:1000; #ab280888, Abcam, Cambridge, UK), p-Smad3 (1:1000; #ab52903, Cambridge, Abcam, UK), LC3 (1:1000; #ab192890, Abcam, Cambridge, UK), Beclin-1 (1:1000; #ab207612, Abcam, Cambridge, UK), P62 (1:1000; #sc-28359, Santa Cruz, CA, USA), α-SMA (1:1000; #55135-1-AP Proteintech, Rosemont, IL, USA), fibronectin (FN) (1:500; #sc-8422, Santa Cruz, CA, USA), and GAPDH (1:1000; #ab8245, Abcam, Cambridge, UK). Subsequently, the membranes were incubated with HRP anti-rabbit IgG (1:2000; #ab288151, Abcam, Cambridge, UK). Finally, the bands were visualized using the ECL- chemiluminescent kit (Proteintech, Rosemont, IL, USA) and quantified using ImageJ software.

Anti-HERC3 (HPA039170, 1:500 dilution), anti-HA (SAB4300603, 1:5000 dilution), and anti-Myc (SAB4301136, 1:2000 dilution) were purchased from Sigma-Aldrich. Anti-EIF5A2 (16386-1-AP, 1:500 dilution), anti-E-Cadherin (20874-1-AP, 1:5000 dilution), N-Cadherin (22018-1-AP, 1:2000 dilution), Vimentin (10366-1-AP, 1:2000 dilution), anti-GAPDH (60004-1-Ig, 1:2000 dilution), anti-Flag (20543-1-AP, 1:5000 dilution), and anti-GST (HRP-66001, 1:5000 dilution) were purchased from Proteintech. Anti-TGF beta1 (ab215715, 1:1000 dilution), antiphospho-Smad2 (phosphor S467) (ab280888, 1:1000 dilution), antiphospho-Smad3 (phosphor S423 + S425) (ab52903, 1:1000 dilution), anti-Smad2 (ab40855, 1:1000 dilution), and anti-Smad3 (ab40854, 1:1000 dilution) were purchased from abcam. The antibodies were used according to the corresponding instructions.

The protein for western blot analysis was extracted from skin tissues and concentration was determined using a BCA protein assay kit (Cat No: FD2001, Hangzhou Fude Biological Technology Company, China). The primary antibody included VEGFR (1:1000, ab39638, Abcam, UK), α-SMA (1:1000, ab124964, Abcam, UK), collagen I (1:1000, ab270993, Abcam, UK), collagen III (1:1000, ab7778, Abcam, UK), TGFβ1 (1:2000, ab215715, Abcam, UK), Smad3 (1:2000, ab40854, Abcam, UK), Smad2 (1:2000, ab40855, Abcam, UK), P-smad3 (phospho T179, 1:2000, ab74062, Abcam, UK), P-smad2 (phospho S467, 1:2000, ab280888, Abcam, UK), TIMP1 (1:2000, ab216432, Abcam, UK), GAPDH (1:4000, 2118, Cell Signaling Technology, Boston, USA) and Anti-rabbit secondary antibody (1:4000, ab6721, Abcam, UK). Proteins were visualized through a Clarity TM Western ECL Substrate (170–5061; Bio-Rad Laboratories Inc., USA) and a Tanon 5200 fully automatic chemiluminescence image analysis system (Tanon Science and Technology Co. Ltd., Shanghai, China).

Tissues and cultured cells were lysed for 30 min with RIPA lysis buffer supplemented with protease inhibitor (Roche, Mannheim, Germany). To analyze inducible protein expression, 20 μg protein was resolved by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted in polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 hour. The separated proteins were then incubated with primary antibodies: anti-GAPDH (CST, #5174, 1:1000), anti-HDAC5 (Invitrogen, PA1-41117, 1:1000), anti-Col1a1 (CST, #72026, 1:1000), anti-Col3a1 (NOVUS, NBP1-05119, 1:500), anti-Smad2 (Abcam, ab33875, 1:1000), anti-p-Smad2 (Abcam, ab280888, 1:1000), anti-Smad3 (Abcam, ab208182, 1:1000), anti-p-Smad3 (Abcam, ab52903, 1:2000), anti-Smad4 (Abcam, ab40759, 1:5000), anti-Smad6 (Abcam, ab273106, 1:1000), anti-Smad7 (Santa Cruz, sc-365846, 1:1000), anti-TGFβRI (Abcam, ab235578, 1:1000), anti-TGFβRII (Abcam, ab259360, 1:1000), anti-Gremlin-1 (Santa Cruz, sc-515877, 1:500), anti-MEF2A (Santa Cruz, sc-17785, 1:1000), anti-Ac-lysine (Santa Cruz, sc-32268, 1:500) antibodies at 4 °C overnight. The membranes were incubated with peroxidase-conjugated secondary antibody at room temperature on the next day. Quantitative analysis was performed on the immunoreactive bands with Image J software.