Cleaved caspase 3

Protein expression levels of Bax, Bcl-2, and cleaved caspase 3 (Wanlei Biotechnology, Shenyang, China) in renal tissues were determined by western blot analysis. Briefly, frozen kidney tissues were lysed with radioimmunoprecipitation assay lysis buffer mixed with phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). After detection of total protein concentrations with a bicinchoninic acid assay kit (Beyotime Biotechnology), samples with equal volumes of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with primary antibodies of Bax (1 : 1000), Bcl-2 (1 : 500), and cleaved caspase 3 (1 : 1000) in Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4°C. After washing, membranes were incubated with goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) at 37°C for 2 h. All protein bands were captured with Amersham Imager 600 software (GE, Boston, MA, USA) and quantified with ImageJ.

Antibodies targeting the following proteins were used in this study: cyclin D1 (WL01435a), CDK2 (WL01543), p27 (WL04174), cleaved caspase-9 (WL01838), cleaved caspase-3 (WL01992), Bcl-2 (WL01556), Bax (WL01637), LC3BII/I (WL01506), beclin-1 (WL02508), p62 (WL02385), p-JNK (WL01813), JNK (WL01295), c-JUN (WL02863), E-cadherin (WL01482), vimentin (WL01960), MMP-2 (WL1579), MMP-9 (WL01580), β-actin (WL01372), goat anti-rabbit IgG-HRP (WLA023) (all from Wanleibio, China), and p-c-JUN (AF3095, Affinity, United States). Purified peiminine (98%) was obtained from Yuanye Bio Co., Ltd (Shanghai, China; B20081). Cell Counting Kit-8 (CCK-8; C0037), the live/dead assay kit, the cell cycle and apoptosis analysis kit, the ROS Kit, NAC, SP600125, and the terminal dUTP nick-end labeling (TUNEL) Kit (C1086) were acquired from Beyotime Institute of Biotechnology (Shanghai, China). Tetramethylrhodamine methyl ester (TMRM; I34361) and Hoechst 3342 (H3570) were sourced from Life Technology (OR, United States). The mRFP-GFP-LC3 adenovirus was acquired from HanBio Technology Co., Ltd (Shanghai, China). The rest of the reagents and experimental materials were purchased from common commercial sources.

CTD (purity ≥98%, product number: MB6525) was purchased from Dalian Meilun Biotechnology Co., Ltd. and added with an appropriate amount of DMSO solution to dissolve into a 32 mM stock solution, stored at −20°C for further use. The antibodies against caspase 3, cleaved caspase 3, cleaved caspase 8, Cyclin E, Cyclin B1, CDK2, p21, p27, and p53 were obtained from Wanlei Biotechnology (Shenyang, China). The antibodies against Nur77, PARP, cleaved PARP, c-Jun, Jun-B, Bcl-2, and Bax were purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-actin, mouse, or rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hoechst 33342 was obtained from Wanlei Biotechnology (Shenyang, China). Other chemicals were obtained from Sigma-Aldrich, unless indicated otherwise.

Total protein from cells or tissues was extracted and quantified. Twenty‐five micrograms of proteins was separated by 10% polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane. Skim milk (5%) was used to block non‐specific antigen binding, and then primary antibodies were added (RASSF1A, ab23950, Abcam; YAP, #14074, Cell Signaling Technology; pS127‐YAP, #13008; Cell Signaling Technology; Cyclin A, sc‐596, Santa Cruz Biotechnology; Cyclin B1, 55004‐1‐AP, Proteintech; Cyclin D1, sc‐246, Santa Cruz Biotechnology; Cyclin E, sc‐25303, Santa Cruz Biotechnology; CDK1, WL02373, Wanleibio; cleaved‐caspase‐3, WL02348, Wanleibio; Bcl‐2, WL01556, Wanleibio; BAX, 50599‐2‐lg, Proteintech; p73, WL01604, Wanleibio; p53, sc‐126, Santa Cruz Biotechnology; p21, WL0362, Wanleibio; AKT, WL0003b, Wanleibio; p‐AKT, WLP001a, Wanleibio; ERK, WL01864, Wanleibio; p‐ERK, WLP1512, Wanleibio; STAT3, WL03207, Wanleibio; p‐STAT3, WLP2412, Wanleibio; NF‐κB P65, WL01980, Wanleibio; LATS1/2, YT2543, ImmunoWay Biotechnology; p‐LATS1/2, YP1222, ImmunoWay Biotechnology; MST1/2, 37462, Signalway Antibody; p‐MST1/2, bs‐3294R, Bioss; β‐actin, sc‐47778, Santa Cruz Biotechnology; Histone H3, Wanleibio) and incubated at 4°C overnight. Next, secondary antibodies were added, followed by incubation at room temperature for 1 hour and subsequent exposure to an X‐ray film.