Ab63856

For immunoblotting, proteins were isolated from cells using RIPA buffer. Total protein extracts (15–50 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane. Membranes were then probed with anti-bax (1:2000; #ab32503; Abcam), bcl-2 (1:2000; #ab196495; Abcam), cleaved-caspase-3/caspase-3 (1:2000; #ab184787; Abcam), SM22α (1:2000; #ab14106; Abcam), calponin (1:500; #ab227661; Abcam), SMMHC (1:2000; #ab125884; Abcam), vimentin (1:2000; #ab92547; Abcam), collagen I (1:1000; #ab270993; Abcam), osteopontin (OPN; 1:1000; #ab63856; Abcam), LAMC1 (1:1000; #ab233389; Abcam), EVI1 (1:1000; #SAB2100723; Sigma), AKT3 (1:2000; #ab152157; Abcam), TP53INP1 (1:2000; #ab202026; Abcam), and β-actin (1:2000; #ab8226; Abcam) at room temperature for 1.5 h. Then, membranes were immersed with the HRP-conjugated secondary antibody at room temperature for 1 h. Following this, the BM chemiluminescence blotting system (Thermo Scientific) was used for detection and protein bands were quantified using Image J software (NIH, USA).

Naringenin (Nar) (≥ 95%, N5893), streptozotocin (STZ) were purchased from Sigma (Sigma Aldrich, United States). Anti-PCNA (1:1000, ab29) and anti-OPN (1:500, ab63856) were purchased from Abcam. Anti-MMP2 (1:1000, bs-4605R) and anti-MMP9 (1:1000, bsm-54040R) were obtained from Bioss Biotechnology Co., LTD. (Beijing, China). Anti-α-SMA (1:1000, BM0002), anti-VEGFA (1:1000, BA0407) and anti-VEGFR2/KDR (1:1000, A00901-3) were purchased from Boster Biological Technology Co. Ltd. (Shanghai, China). Anti-CREB5 (1:1000, G420) was purchased from Santa cruz. The following antibodies: PI3K (1:1000, 4249), p-PI3K (1:500, 17366), Akt-pan (1:1000, 4685), p-Akt (Ser 473) (1:1000, 4060), Src (1:1000, 36D10), and p-Src (1:1000, D49G4) were obtained from CST. EdU Imaging kit was purchased from APE × Bio (United States). Trypsin, Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO.

Immunohistochemistry detection of Rho A, ROCK1, ROCK2, GLUT1, HK2, PDK1, PFK1, LDHA, OPN and RUNX2 in human aortic valve leaflets was performed. 4% PFA-fixed paraffin sections (5μm thick) were deparaffinized using dewaxed solution and hydrated using decreasing concentration of ethyl alcohol (100%, 90%, 80%, 70%, 60%), and then incubated at 65 °C for 20 min in antigen unmasking solution using PT Module-Lab Vision (Thermo Fisher Scientific) for antigenic retrieval. Following several washes (3 x 5 min) in PBS, paraffin sections were blocked for 30 min using 5% bovine serum albumin (BSA). After blocking, primary antibodies against Rho A (abcam, ab54835), ROCK1 (abcam, ab97592), ROCK2 (abcam, ab125025), GLUT1 (abcam, ab115730), HK2(abcam, ab209847), PDK1 (abcam, ab202468), PFK1 (CST, #8164), LDHA (abcam, ab52488), OPN (abcam, ab63856) and RUNX2 (abcam, ab192256) were diluted at optimized concentrations in 1% BSA and incubated with the sections in a wet box overnight at 4 °C. Subsequently, appropriate secondary antibodies were applied for 30 min at room temperature. Following secondary antibody incubation, the positive staining was detected using an UltraSensitive SP IHC Kit and DAB Kit, and hematoxylin staining was used for nuclear counterstaining. Images were visualized using a microscope (CKX53, Olympus), and Image J software was applied for data quantification.

Cholecalciferol (vitamin D3) (C9756, Sigma Aldrich, St. Louis, MO, USA). Mucolipin1 (SC-26269, Dallas, TX, USA), OSP (ab63856, Abcam, Cambridge, MA, USA), RUNX2 (ab23981, Abcam, Cambridge, MA, USA), SM22-α (ab14106, Abcam, Cambridge, MA, USA), VPS16 (Cat. No.17776–1-AP, Protein biotech group, Rosemont, IL, USA), Rab7 (ab137029, Abcam, Cambridge, MA, USA), ALG-2 (PDCD6, A6685, Cummings Park Dr, Woburn, MA, USA.), CD63 (ab216130, Abcam, Cambridge, MA, USA) and annexin-II (AnX2, ab41803, Abcam, Cambridge, MA, USA). Secondary antibodies are Alexa-488 or Alexa-555-labeled (Life technologies, Grand Island, NY, USA). Rat monoclonal anti-mouse Lamp-1 (ab25245, Abcam, Cambridge, MA, USA). Alizarin Red S Solution (TMS-008-C, EMD Millipore. Burlington, MA, USA) was used for detection of arterial medial calcification.

To conduct cell protein immunodetection, Western-blot (WB) assay was conducted for determining osteogenic protein expression within rBMSCs. Total Protein Extraction Reagent (Aspen) was utilized for cellular protein separation on ice. Proteins were boiled within the loading buffer for an 8-min period under 99 °C, then protein aliquots were exposed to 12% SDS-PAGE for separation, followed by transfer on PVDF membranes (Millipore). Primary antibodies against RUNX2 (Abcam) and ALP (LSBio) were added to incubate membranes on day 5, whereas those against OCN (sc-390,877, 1:500, Santa Cruz) and OPN (ab63856, 1:1000, Abcam) were added to incubate membranes on day 14. Later, secondary antibodies (1:10000) were added to further probe membranes, followed by detection with chemiluminescence (ECL) (AS1059, Aspen). In addition, the ChemiDoc Touch chemiluminescent system (Biorad) was applied in protein visualization. GAPDH (ab181602, Abcam) served as the reference for normalizing osteogenic marker levels. According to gray value, image J was utilized to analyze target protein expression.