3 methyladenine

CCECs at passage 2 were randomly divided into five groups: (1) control (homoculture of CCECs only); (2) bovine serum albumin (BSA; Abcam, USA); (3) AGE-BSA (AGEs; Abcam); (4) AGEs+USCs; and (5) AGEs+USCs+3-methyladenine (3-MA; Selleckchem, USA).
The coculture was performed in a Transwell unit (Costar 3413, Corning, USA). Cell suspensions of CCECs or USCs were prepared in endothelial cell growth media, respectively, at the concentration of 1 × 10⁵ cells/ml. CCECs (600 μl) were cultured in the lower chamber, and USCs (100 μl) were cultured in the upper chamber separately. After being incubated overnight, CCECs were infected with mRFP-GFP-LC3 adenovirus (HanBio Technology, Shanghai, China) according to the instructions. After 2 h, CCECs were changed with complete medium, and BSA/AGEs was added into the culture medium according to the grouping for 72 h. For groups (4) and (5), the upper chambers with USCs were placed above the lower CCEC chambers. For group (5), 3-MA was added into the lower chambers for 24 h. All experiments were performed in triplicate. The concentration of AGEs (200 μg/ml) and 3-MA (2 mM) used in the present study was referred to previous studies [33 (link)–35 (link)].

After PEG-mediated transformation, the rice protoplasts were placed in 1 ml W5 buffer containing 10 μM 3-MA (3-methyladenine, Selleck, S2767), 100 μM chloroquine (MCE, HY-17589A), 100 μM leupeptin (MCE, HY-18234A), or 20 μM E-64d (Aloxistatin, Sigma-Aldrich, E8640) to inhibit autophagy or 50 μM MG132 (Selleck, S2619) to inhibit the 26S proteasome. The treated protoplasts were incubated at 28 °C for 14–20 h before protein extraction. Anti-actin (Abbkine, ABL1050) was used as a reference to quantify total protein levels. Rice WRKY72 is degraded by the 26S proteasome pathway^{19 (link)} and was used as a positive control to ensure that the proteasome inhibitor MG132 was active. The rice HD1 protein is degraded in the dark by the autophagy pathway^{61 (link)}, and OsNBR1 was used as a positive control to assure autophagy inhibitors were active. The intensity of protein signals in immunoblots was quantified by ImageJ.

Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FBS) and Dulbecco’s Modified Eagle’s Medium were purchased from HyClone (Logan, UT, USA). The autophagy inhibitor, 3-Methyladenine, was ordered from Selleck, USA. The small interfering RNA of AMPK (sc-45312) and normal control (sc-37007) came from Santa Cruz (CA, USA). BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075s) was obtained from Beyotime Biotechnogy (Shanghai, China). The primary antibody of LC3B (L7543) for Western blot was obtained from Sigma-Aldrich (St. Louis, MO, USA). Other primary antibodies of cyclinD1 (AF0126), PCNA (AF1363), ERK (AF1051), p-ERK (AF5818), p62/SQSTM1 (AF5312), AMPKα (AF6195), and p-AMPK (Thr172) (AF5908) were ordered from Beyotime Biotechnology (Shanghai, China). The primary anti-LC3B antibody with Alexa Flour 647 fluorescence for immunofluorescence was from Abcam (Cambridge, MA, USA). The antibodies of LC3B (GB13431) and Ki67 (GB111499) for immunohistochemistry were attained from Servicebio (Wuhan, China). All the antibodies were used at a ratio of 1:1000 in Western blot assays, and p-AMPK (1:50), LC3B (1:300) and Ki-67 (1:600) were used in Immunohistochemistry assays.