P27kip1

The in vitro kinase assay was performed by EnzyChrom Kinase Assay Kit (MEDIBENA Life Science & Diagnostic Solutions, Vienna, Austria) according to manufactures’ instruction. Briefly, set up 75 μl reaction mixture containing recombinant CDK5/p25 (Invitrogen) as kinase, ATP and recombinant p53 (Millipore, Billerica, MA), p27Kip1 (Abcam, Cambridge, MA), p21Cip1 (Abcam, Cambridge, MA), Histione H1 (Santa Cruz, Santa Cruz, CA) and ARHI NTD (OriGene, Rockville, MD) as substrate in the Assay Buffer, respectively. Incubate at room temperature for 20 min. Aliquot 20 μl mixture into 3 separate wells on 96 well plate, add 40 μl working reagent to each assay well. Incubate at room temperature for 10 min and read fluorescence intensity (excitation = 530nm and emission = 590nm). Calculate kinase activity as the formula below.
δFluorescence = (fluorescence intensity of sample well—blank well) and slope is the slope of the ADP standard curve.

Sections were stained immunohistochemically using antibodies against smooth muscle α-actin (αSMA, 1A4, DAKO), von Willebrand Factor (Millipore) and p27^Kip1 (Abcam), followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Southern Biotech) or poly HRP-anti-rabbit IgG (Immunologic, Duiven, the Netherlands) followed by 3,3-diaminobenzidine (DAB) substrate color development (Immunologic). p27^Kip1 quantification was performed on 3 areas per stent section for all stents and expressed as positive area of the intima.

The pancreases were excised, fixed overnight in 10% paraformaldehyde and embedded in paraffin. Samples were sectioned at 3 μm and stained with hematoxylin-eosin or incubated with primary antibodies: p21Cip1(ab2961, Abcam, Cambridge, UK), p27Kip1(ab7961, Abcam), p57Kip1(ab75974, Abcam), p53 (NCL-p53-CM5p, Leica Biosystems, Wetzlar, Germany), CHOP (sc-575, Santa Cruz Biotechnology, CA, USA), GFP (sc-8334, Santa Cruz), Ki-67 (#12202, Cell Signaling Technology, MA, USA), insulin (I2018, Sigma) or glucagon (A0565, Dako, CA, USA). The immune complexes were visualized with DAB (Histofine Simple Stain Mouse MAX-PO (R) or Histofine Mouse Stain Kit; Nichirei, Tokyo, Japan). Alexa Fluor 488 goat anti-mouse IgG (Sigma) or Alexa Fluor 546 goat anti-rabbit IgG (Dako) was used as the fluorescent secondary antibody. Dapi-Fluoromount-G™ (Southern Biotech, AL, USA) was used to stain nuclei in the final step. At least 20 islets with > 1000 islet cells were counted per mouse for evaluation of p21, p27, p53, CHOP, GNZ and Ki-67 expression by IHC staining. GNZ protein was stained with anti-GFP antibody. Both positive and negative cells were counted manually using the ImageJ Cell Counter plugin.

Proteins were extracted from cells using an ice-cold RIPA lysis solution supplemented with protease and phosphatase inhibitors (Thermo Fisher, USA). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, China) and boiled for 10 min at 100 °C. Protein lysates were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Merck Millipore, USA), and blocked for 1.5 h at room temperature with 5% skim milk. After probing with primary antibodies overnight at 4 °C, membranes were incubated for 2 h at room temperature with HRP-conjugated anti-rabbit or anti-mouse. The Image Lab software was used to enhance the chemiluminescence (ECL, Thermo Scientific, USA) and view protein bands (Bio-Rad, USA). The antibodies used were: PTBP1 (CST, cat. no.: 57246S), c-Myc (Abcam, cat. no.: ab32072), pho-c-Myc^T58 (Abcam, cat. no.: ab185655), pho-c-Myc^S62 (Abcam, cat. no.: ab185656), Nanog (CST, cat. no.: 4903S), Oct4 (CST, cat. no.: 2750S), Sox2 (CST, cat. no.: 3579S), FBW7 (Abcam, cat. no.: ab109617), Lamin A/C (Abcam, cat. no.: ab108595), USP28 (Abcam, cat. no.: ab126604), p21 (Abcam, cat. no.: ab109520), p27^kip1 (Abcam, cat. no.: ab32034), Cyclin D1 (Abcam, cat. no.: ab16663) and β-actin (CST, cat. no.: 4970S).

HASMCs were collected and lysed in RIPA buffer (pH 7·4) containing 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris)-HCl (50 mM), NaCl (150 mM), EDTA (1 mM), Triton-X (1%), sodium deoxycholate (0.5%) and SDS (0.1%).
The homogenates were centrifuged at 13000 r/min for 15 min at 4 °C and the supernatant was obtained. Proteins were fractured by electrophoresis on a 10% SDS-polyacrylamide gel and transferred on onto PVDF membrane (Millipore, USA). Membranes were soaked in 5% milk/PBST for blocking.
Then, membranes were incubated with antibodies to p27kip1 (Abcam, USA), CyclinE (CST, USA), CyclinD1 (CST, USA), PI3K (Abcam, USA), p-PI3K (Abcam, USA), AKT (Abcam, USA), p-AKT (Abcam, USA) overnight at 4 °C. The membrane washed three times in PBST and Hrp-conjucated secondary antibody goat anti-rabbit or goat anti-mouse (Abcam, USA) were added on the membranes for 1 h in room temperature. Protein bands were visualized by staining with enhanced chemiluminescence (ECL).

After being cultured with or without recombinant CTGF (Cloud-Clone Corp, USA)/CTGF-siRNA, the TSPCs were lysed and centrifuged and the supernatant was then collected for the measurement of protein concentration by the BCA protein assay (Thermo Scientific). 30 μg of protein was denatured, fractionated by electrophoresis on SDS-PAGE, and electrophoretically transferred to a PVDF membrane (Millipore, USA). The blots were blocked with 5% nonfat dry milk in PBST solution and incubated with primary antibodies against CTGF (Proteintech, USA), p16^INK4A (Novus Biologicals, USA), p27^KIP1 (Abcam, USA), CDK4 (Proteintech, USA), cyclin D1 (Bioworld, USA), and β-actin (Proteintech, USA) at 4°C overnight. After incubating with a secondary antibody, immunoreactive bands were detected by ECL reagents (Pierce, USA). The gray value of each band was measured, and data are presented as a ratio to β-actin.

Sections were stained immunohistochemically using antibodies against smooth muscle α-actin (1A4, DAKO), macrophages (RAM11, DAKO), cleaved caspase-3 (Asp175, Cell Signaling), PCNA (BD) and p27^Kip1 (Abcam), followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Southern Biotech) or poly HRP-anti-rabbit IgG (Immunologic, Duiven, the Netherlands) followed by 3,3-diaminobenzidine (DAB) substrate color development (Immunologic). Inflammation score according to Kornowski was determined on RAM11-stained sections, allocating values of 0 (none), 1 (mild with minimal infiltrated inflammatory cells), 2 (moderate) or 3 (severe, with large clusters of inflammatory cells with granulomatous morphology) [29 (link)]. Cleaved caspase-3, PCNA and p27^Kip1 quantification was performed on 4 areas per stent section for all stents and expressed as positive area of the neointima. Additionally, the number of adventitial capillaries was counted.