P21cip1

Chromatographically pure Sch was purchased from Feng-Shan-Jian Medicine Research Co. Ltd. (Kunming, Yunnan, China) (Structure shown in Figure 1), and dissolved in 0.5% dimethyl sulfoxide (DMSO) at a concentration of 0.5 mg/mL before experiment. LPS (Escherichia coli serotype O55:B5), Evans blue (EB) dye, and rhodamine 6G were purchased from Sigma Chemical (St. Louis, MO, United States). Primary antibodies against I-κBα, phospho-I-κBα, NF-κB p65, Erk1/2, phospho-Erk1/2, p38 MAPK, phospho-p38 MAPK, phospho-FoxO1, Akt, phospho-Akt, Keratin, caspase-3, cyclin D1, cyclin B1, p27^kip1, phospho-retinoblastoma protein (Rb), GAPDH, and histone H3 were obtained from Cell Signaling Technology (Beverly, MA, United States). Antibodies against Claudin-5, Occludin and ZO-1 were from Invitrogen (Rochford, IL, United States), antibodies against FoxO1, p21^cip1, myeloperoxidase (MPO) and von Willebrand factor (vWF) from Abcam (Cambridge, United Kingdom). Antibody against VE-Cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, United States), antibody against TLR4 from Novus Biologicals (Littleton, CO, United States) and antibody against prosurfactant protein C (proSP-C) from EMD Millipore Corporation (Temecula, CA, United States).

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.

Terminal liver samples were dissected from the left lateral lobe immediately after killing the animal and subsequently fixed overnight in 4% paraformaldehyde. The liver tissue was then paraffin-embedded and sectioned at a thickness of 3 µm. Sections were stained with H&E (Dako), or DAPI (Invitrogen) co-stained with p21^Cip1 (ab188224, Abcam). Slides were scanned by ScanScope AT System (Aperio).

Primary antibodies were obtained for N-MYC from Santa Cruz (Dallas, TX). E-cadherin antibodies were from Cell Signaling Technology (Danvers, MA), and p21/Cip1 and PARP primary antibodies were obtained from Abcam (Cambridge, MA). All other reagents were obtained from Sigma (St. Louis, MO).