Sc 7907

Primary human CD34⁺ cells, AML cell lines transduced with eGFP and GFI1-eGFP lentiviral vectors were sorted and nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Darmstadt, Germany). Antibodies used for Western blotting included goat polyclonal antibody against GFI1 (N-20) (1:1000, G6670, Sigma-Aldrich, Darmstadt, Germany), rabbit polyclonal antibody against PCNA (1:500, sc-7907, Santa Cruz Biotechnologies, Minneapolis, USA), donkey anti-goat IgG-HRP (1:5000, sc-2020, Santa Cruz Biotechnologies) and Goat anti-rabbit IgG-HRP (1:2500, sc-2030, Santa Cruz Biotechnologies). ECL Western blot analysis system (Amersham) was used for detection.

Creatinine and Urea plasma levels were assessed by biochemical methods intended for automatic measurements in a biochemistry analyzer (Roche/Hitachi cobas® c701/702) based on the enzymatic decomposition with creatininasa and ureasa, respectively, then followed by colorimetric detection of the reactions products.
Tubular injury was assessed in kidney tissue sections stained with hematoxylin–eosin by a renal pathologist (P.C) blinded as to the nature of the samples. Evidence of tubulointerstitial injury including pyknosis/apoptosis, activity, proliferation/regeneration, flattening, necrosis, calcifications and intratubular precipitations were individually scored on a semiquantitative scale from 0 to 3. Results from each item were added to yield the overall tubular injury score (maximal value 21) as previously described²⁹ .
For immunohistochemistry, endogenous peroxidase was blocked and then sections were incubated overnight at 4 °C with the following primary antibodies: PCNA (1:50; sc-7907, Santa Cruz); active caspase-3 (1/100, G748A, Promega); phospho-cJUN (1/250, 3270, Cell Signaling) or 4-hydroxynonenal (4-HNE) (1:400; ab46545; Abcam). Finally, sections were washed, stained with 3,3′-diaminobenzidine (DAB) as chromogen (Dako, Denmark), counterstained with Carazzi’s hematoxylin, dehydrated and mounted in DPX medium (Merck, NJ, USA).

Cell lysates were separated by SDS PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed by using primary antibodies against ASPH; JAG1, JAG2, DLL1, DLL4, Notch1, Notch2, Notch3, Notch4, c-Myc, matrix metalloproteinase (MMP)-2, epithelial cell adhesion molecule (EpCAM), and CD44 (Cell Signaling Technology #2620, #2205, #2588, #2589, #4380, #4530, #5276, #2423, #5605, #13132, #2929, #5640, respectively); activated Notch1, HES1 and HEY1 (Abcam ab8925, ab71559, ab22614, respectively); cyclin D3, MMP9, and proliferating cell nuclear antigen (PCNA) (Santa Cruz, sc-56307, sc-21733, sc-7907, respectively). Protein bands were visualized by IRDye® 680RD Infrared Dye and IRDye® 800CW Infrared Dye and exposed on Odyssey image system (LI-COR).

For all experimental groups, formalin-fixed and paraffin-embedded colonic mucosa sections were stained with hematoxylin and eosin for conventional histopathological analysis. Immunohistochemical staining for PCNA and β-catenin were performed using the labeled streptavidin-biotin method (LSAB kit; Dako, Glostrup, Denmark), as previously described [14 (link),24 (link),25 (link)]. The primary antibodies for β-catenin and PCNA were obtained from BD Transduction Laboratories (No. 610154; San Jose, CA, USA) and Santa Cruz Biotechnology (sc-7907; Santa Cruz, CA, USA), respectively. PCNA-positive cells in the colonic mucosa were counted and expressed as a percentage of the total number of normal crypt cells. The PCNA labeling index (%) was determined from the assessment of a minimum of 200 crypt cells in each mouse [29 (link)].

1.5 × 10⁶ of 293T cells infected with GIPZ-shTMED3 or control lentiviruses were seeded in 6-cm dishes in DMEM with 10% FBS. The following day the infected cells were transfected using calcium phosphate with 0.8 μg of V5-tagged WNT3A expression plasmid (Addgene 35927). Medium was replaced 6 h after by 4 ml of DMEM with 0.5% FBS. Supernatants were collected 48 h later, spun twice on a table top centrifuge to remove any floating cells or debris and concentrated ∼20-fold (Pierce Concentrators 9K MWCO, 89884A, Thermo Scientific). Cells were also collected directly from the dish and lysed in RIPA buffer. 25 μg of total protein from the concentrated supernatants or 2 μg from the cell lysates were loaded per well onto 12% SDS–PAGE. Western blotting was performed using anti-V5 (ab27671, Abcam, 1/500 overnight) and anti-PCNA (SC-7907, Santa Cruz, 1/1,000 1 h) antibodies.

The primary antibody for PCNA was purchased from Santa Cruz Biotechnology (sc-7907, Santa Cruz, CA, USA). The primary antibody for CD31 was purchased from Abcam (ab28364, Abcam, Cambridge, United Kingdom). According to instructions of the Envision System-HRP method (DakoCytomation Inc, Carpinteria, CA, USA), tumor sections were stained.